Arrays of nucleic acid probes for analyzing biotransformation genes

ABSTRACT

The invention provides arrays of immobilized probes, and methods employing the arrays, for detecting mutations in the biotransformation genes, such as cytochromes P450. For example, one such array comprises four probe sets. A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence from a biotransformation gene, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence. Second, third and fourth probe sets each comprise a corresponding probe for each probe in the first probe set. The probes in the second, third and fourth probe sets are identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 09/798,260,filed on Mar. 1, 2001, which is a continuation of U.S. Ser. No.08/778,794, filed on Jan. 3, 1997, now U.S. Pat. No. 6,309,823, which isa continuation in part of U.S. Ser. No. 08/544,381, filed on Oct. 10,1995, now U.S. Pat. No. 6,027,880, which is a continuation in part ofU.S. Ser. No. 08/510,521, filed Aug. 2, 1995, which is acontinuation-in-part of PCT/US94/12305, filed Oct. 26,1994,-which is acontinuation in part of U.S. Ser. No. 08/284,064, filed Aug. 2,1994,which is a continuation in part of U.S. Ser. No. 08/143,312, filed Oct.26, 1993, each of which is incorporated by reference in its entirety forall purposes. Research leading to the invention was funded in part byNIH grant No.1R01HG00813-01, and the government may have certain rightsto the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides arrays of oligonucleotide probesimmobilized in microfabricated patterns on chips for analyzingbiotransformation genes, such as cytochromes P450.

2. Description of Related Art

Virtually all substances introduced into the human body (xenobiotics) aswell as most endogenous compounds (endobiotics) undergo some form ofbiotransformation in order to be eliminated from the body. Many enzymescontribute to the phase I and phase II metabolic pathways responsiblefor this bioprocessing. Phase I enzymes include reductases, oxidases andhydolases. Among the phase I enzymes are the cytochromes P450, asuperfamily of hemoproteins involved in the oxidative metabolism ofsteroids, fatty adds, prostaglandins, leukotrienes, biogenic amines,pheromones, plant metabolites and chemical carcinogens as well as alarge number of important drugs (Heim & Meyer, Genomics 14, 49-58(1992)). Phase II enzymes are primarily transferases responsible fortransferring glucuronic acid, sulfate or glutathione to compoundsalready processed by phase I enzymes (Gonzales & Idle, Clin.Pharmacokinet. 26, 59-74 (1994)). Phase II enzymes include epoxidehydrolase, catalase, glutathione peroxidase, superoxide dismutase andglutathione S-transferase.

Many drugs are metabolized by biotransformation enzymes. For some drugs,metabolism occurs after the drug has exerted its desired effect, andresult in detoxification of the drug and elimination of the drug fromthe body. Similarly, the biotransformation enzymes also have roles indetoxifying harmful environmental compounds. For other drugs, metabolismis required to convert the drug to an active state before the drug canexert its desired effect.

Genetic polymorphisms of cytochromes P450 and other biotransformationenzymes result in phenotypically-distinct subpopulations that differ intheir ability to perform biotransformations of particular drugs andother chemical compounds. These phenotypic distinctions have importantimplications for selection of drugs. For example, a drug that is safewhen administered to most human may cause intolerable side-effects in anindividual suffering from a defect in an enzyme required fordetoxification of the drug. Alternatively, a drug that is effective inmost humans may be ineffective in a particular subpopulation because oflack of a enzyme required for conversion of the drug to a metabolicallyactive form. Further, individuals lacking a biotransformation enzyme areoften susceptible to cancers from environmental chemicals due toinability to detoxify the chemicals. Eichelbaum et al., ToxicologyLetters 64/65, 155-122 (1992). Accordingly, it is important to identifyindividuals who are deficient in a particular P450 enzyme., so thatdrugs known or suspected of being metabolized by the enzyme are notused, or used only with special precautions (e.g., reduced dosage, closemonitoring) in such individuals. Identification of such individuals isalso important so that such individuals can be subjected to regularmonitoring for the onset of cancers.

Existing methods of identifying deficiencies are not entirelysatisfactory. Patient metabolic profiles are currently assessed with abioassay after a probe drug administration. For example, a poor drugmetabolizer with a CYP2D6 defect is identified by administering one ofthe probe drugs, debrisoquine, sparteine or dextromethorphan, thentesting urine for the ratio of unmodified to modified drug. Poormetabolizers (PM) exhibit physiologic accumulation of unmodified drugand have a high metabolic ratio of probe drug to metabolite. Thisbioassay has a number of limitations: lack of patient cooperation,adverse reactions to probe drugs, and inaccuracy due to coadministrationof other pharmacological agents or disease effects. Genetic assays byRFLP (restriction fragment length polymorphism), ASO PCR (allelespecific oligonucleotide hybridization to PCR products or PCR usingmutant/wildtype specific oligo primers), SSCP (single strandedconformation polymorphism) and TGGE/DGGE (temperature or denaturinggradient gel electrophoresis), MDE (mutation detection electrophoresis)are time-consuming, technically demanding and limited in the number ofgene mutation sites that can be tested at one time.

The difficulties inherent in previous methods are overcome by the use ofDNA chips to analyze mutations in biotransformation genes. Thedevelopment of VLSIPS™ technology has provided methods for making verylarge arrays of oligonucleotide probes in very small areas. See U.S Pat.No. 5,143,854, WO 90/15070 and WO 92/10092, each of which isincorporated herein by reference. U.S. Ser. No. 08/082,937, filed Jun.25, 1993, describes methods for making arrays of oligonucleotide probesthat can be used to provide the complete sequence of a target nucleicacid and to detect the presence of a nucleic acid containing a specificnucleotide sequence. Others have also proposed the use of large numbersof oligonucleotide probes to provide the complete nucleic acid sequenceof a target nucleic acid but failed to provide an enabling method forusing arrays of immobilized probes for this purpose. See U.S. Pat. No.5,202,231, U.S. Pat. No. 5,002,867 and WO 93/17126.

Microfabricated arrays of large numbers of oligonucleotide probes,called “DNA chips” offer great promise for a wide variety ofapplications. The present application describes the use of such chipsfor inter alia analysis of the biotransformation genes, such ascytochromes P450.

SUMMARY OF THE INVENTION

The invention provides arrays of probes immobilized on a solid supportfor analyzing biotransformation genes. In a first embodiment, theinvention provides a tiling strategy employing an array of immobilizedoligonucleotide probes comprising at least two sets of probes. A firstprobe set comprises a plurality of probes, each probe comprising asegment of at least three nucleotides exactly complementary to asubsequence of a reference sequence from a biotransformation gene, thesegment including at least one interrogation position complementary to acorresponding nucleotide in the reference sequence. A second probe setcomprises a corresponding probe for each probe in the first probe set,the corresponding probe in the second probe set being identical to asequence comprising the corresponding probe from the first probe set ora subsequence of at least three nucleotides thereof that includes the atleast one interrogation position, except that the at least oneinterrogation position is occupied by a different nucleotide in each ofthe two corresponding probes from the first and second probe sets. Theprobes in the first probe set have at least two interrogation positionscorresponding to two contiguous nucleotides in the reference sequence.One interrogation position corresponds to one of the contiguousnucleotides, and the other interrogation position to the other. In this,and other forms of array, biotransformation genes of particular interestfor analysis include cytochromes P450, particularly 2D6 and 2C19,N-acetyl transferase II, glucose 6-phosphate dehydrogenase,pseudocholinesterase, catechol-O-methyl transferase, and dihydropyridinedehydrogenase.

In a second embodiment, the invention provides a tiling strategyemploying an array comprising four probe sets. A first probe setcomprises a plurality of probes, each probe comprising a segment of atleast three nucleotides exactly complementary to a subsequence of areference sequence from a biotransformation gene, the segment includingat least one interrogation position complementary to a correspondingnucleotide in the reference sequence. Second, third and fourth probesets each comprise a corresponding probe for each probe in the firstprobe set. The probes in the second, third and fourth probe sets areidentical to a sequence comprising the corresponding probe from thefirst probe set or a subsequence of at least three nucleotides thereofthat includes the at least one interrogation position, except that theat least one interrogation position is occupied by a differentnucleotide in each of the four corresponding probes from the four probesets.

In a third embodiment, the invention provides arrays comprising firstand second groups of probe sets, each group comprising first, second andoptionally, third and fourth probe sets as defined above. The firstprobe sets in the first and second groups are designed to be exactlycomplementary to first and second reference sequences. For example, thefirst reference can include a site of mutation rendering the genenonfunctional, and the second reference sequence can include a site of asilent polymorphism.

In a fourth embodiment, the invention provides a block ofoligonucleotides probes (sometimes referred to as an optiblock)immobilized on a support. The array comprises a perfectly matched probecomprising a segment of at least three nucleotides exactly complementaryto a subsequence of a reference sequence from a biotransformation gene,the segment having a plurality of interrogation positions respectivelycorresponding to a plurality of nucleotides in the reference sequence.For each interrogation position, the array further comprises threemismatched probes, each identical to a sequence comprising the perfectlymatched probe or a subsequence of at least three nucleotides thereofincluding the plurality of interrogation positions, except in theinterrogation position, which is occupied by a different nucleotide ineach of the three mismatched probes and the perfectly matched probe.

In a fifth embodiment (sometimes referred to as deletion tiling), theinvention provides an array comprising at least four probes. A firstprobe comprises first and second segments, each of at least threenucleotides and exactly complementary to first and second subsequencesof a reference sequence-from a biotransformation gene, the segmentsincluding at least one interrogation position corresponding to anucleotide in the reference sequence, wherein either (1) the first andsecond subsequences are noncontiguous, or (2) the first and secondsubsequences are contiguous and the first and second-segments areinverted relative to the complement of the first and second subsequencesin the reference sequence. The array further comprises second, third andfourth probes, identical to a sequence comprising the first probe or asubsequence thereof comprising at least three nucleotides from each ofthe first and second segments, except in the at least one interrogationposition, which differs in each of the probes.

In a sixth embodiment, the invention provides a method of comparing atarget nucleic acid with a reference sequence from a biotransformationgene. The method comprises hybridizing a sample comprising the targetnucleic acid to one of the arrays of oligonucleotide probes describedabove. The method then determines which probes, relative to one another,specifically bind to the target nucleic acid, the relative specificbinding of corresponding probes indicating whether a nucleotide in thetarget sequence is the same or different from the correspondingnucleotide in the reference sequence.

For example, for the array of the second embodiment which has four probesets, the array can be analyzed by comparing the relative specificbinding of four corresponding probes from the first, second, third andfourth probe sets, assigning a nucleotide in the target sequence as thecomplement of the interrogation position of the probe having thegreatest specific binding, and repeating these steps until eachnucleotide of interest in the target sequence has been assigned.

In some methods, the reference sequence includes a site of a mutation inthe biotransformation gene and a silent polymorphism in or flanking thebiotransformation gene, and the target nucleic acid comprises one ormore different alleles of the biotransformation gene. In this situation,the relative specific binding of probes having an interrogation positionaligned with the silent polymorphism indicates the number of differentalleles and the relative specific binding of probes having aninterrogation position aligned with the mutation indicates whether themutation is present in at least one of the alleles.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Basic tiling strategy. The figure illustrates the relationshipbetween an interrogation position (I) and a corresponding nucleotide (n)in the reference sequence, and between a probe from the first probe setand corresponding probes from second, third and fourth probe sets.

FIG. 2: Segment of complementarity in a probe from the first probe set.

FIG. 3A: Incremental succession of probes in a basic tiling strategy.The figure shows four probe sets, each having three probes. Note thateach probe differs from its predecessor in the same set by theacquisition of a 5′ nucleotide and the loss of a 3′ nucleotide, as wellas in the nucleotide occupying the interrogation position.

FIG. 3B: Arrangement of probe sets in tiling arrays lacking a perfectlymatched probe set.

FIG. 4A: Exemplary arrangement of lanes on a chip. The chip shows fourprobe sets, each having five probes and each having a total of fiveinterrogation positions (I1-I5), one per probe.

FIG. 4B: A tiling strategy for analyzing closing spaced mutations.

FIG. 4C: A tiling strategy for avoiding loss of signal due to probeself-annealing.

FIG. 5: Hybridization pattern of chip having probes laid down in lanes.Dark patches indicate hybridization. The probes in the lower part of thefigure occur at the column of the array indicated by the arrow when theprobes length is 15 and the interrogation position 7.

FIG. 6: Strategies for detecting deletion and insertion mutations. Basesin brackets may or may not be present.

FIG. 7: Block tiling strategy. The perfectly matched probe has threeinterrogation positions. The probes from the other probe sets have onlyone of these interrogation positions.

FIG. 8: Multiplex tiling strategy. Each probe has two interrogationpositions.

FIG. 9: Helper mutation strategy. The segment of complementarity differsfrom the complement of the reference sequence at a helper mutation aswell as the interrogation position.

FIG. 10: Layout of probes on chip for analysis of cytochrome P450 2D6and cytochrome P450 2C19.

FIG. 11: Alternative tiling for analysis of CyP2D6/CYP2D7 polymorphism.FIG. 12: Optiblock for analysis of CYP2D6P34S polymorphism.

FIG. 13: The chip shown in FIG. 10 hybridized to a CYP2D6-B target.

FIG. 14: Magnification of the hybridization patterns of the cytochromeP450 2D6 L421P and S486 polymorphism opti-tiling blocks.

FIG. 15: Hybridization of the chip shown in FIG. 10 to cytochrome P4502C19.

FIG. 16: VLSIPS™ technology applied to the light directed synthesis ofoligonucleotides. Light (hv) is shone through a mask (M₁) to activatefunctional groups (—OH) on a surface by removal of a protecting group(X). Nucleoside building blocks protected with photoremovable protectinggroups (T-X, C-X) are coupled to the activated areas. By repeating theirradiation and coupling steps, very complex arrays of oligonucleotidescan be prepared.

FIG. 17: Use of the VLSIPS™ process to prepare “nucleosidecombinatorials” or oligonucleotides synthesized by coupling all fournucleosides to form dimers, trimers, and so forth.

FIG. 18: Deprotection, coupling, and oxidation steps of a solid phaseDNA synthesis method.

FIG. 19: An illustrative synthesis route for the nucleoside buildingblocks used in the VLSIPS™ method.

FIG. 20: A preferred photoremovable protecting group, MeNPOC, andpreparation of the group in active form. FIG. 21: Detection system forscanning a DNA chip.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a number of strategies for comparing apolynucleotide of known sequence (a reference sequence) with variants ofthat sequence (target sequences). The comparison can be performed at thelevel of entire genomes, chromosomes, genes, exons or introns, or canfocus on individual mutant sites and immediately adjacent bases. Thestrategies allow detection of variations, such as mutations orpolymorphisms, in the target sequence irrespective whether a particularvariant has previously been characterized. The strategies both definethe nature of a variant and identify its location in a target sequence.

The strategies employ arrays of oligonucleotide probes immobilized to asolid support. Target sequences are analyzed by determining the extentof hybridization at particular probes in the array. The strategy inselection of probes facilitates distinction between perfectly matchedprobes and probes showing single-base or other degrees of mismatches.The strategy usually entails sampling each nucleotide of interest in atarget sequence several times, thereby achieving a high degree ofconfidence in its identity. This level of confidence is furtherincreased by sampling of adjacent nucleotides in the target sequence tonucleotides of interest. The present tiling strategies result insequencing and comparison methods suitable for routine large-scalepractice with a high degree of confidence in the sequence output.

I. General Tiling Strategies

A. Selection of Reference Sequence

The chips are designed to contain probes exhibiting complementarity toone or more selected reference sequence whose sequence is known. Thechips are used to read a target sequence comprising either the referencesequence itself or variants of that sequence. Target sequences maydiffer from the reference sequence at one or more positions but show ahigh overall degree of sequence identity with the reference sequence(e.g., at least 75, 90, 95, 99, 99.9 or 99.99%). Any polynucleotide ofknown sequence can be selected as a reference sequence. Referencesequences of interest include sequences known to include mutations orpolymorphisms associated with phenotypic changes having clinicalsignificance in human patients. For example, the CFTR gene and P53 genein humans have been identified as the location of several mutationsresulting in cystic fibrosis or cancer respectively. Other referencesequences of interest include those that serve to identify pathogenicmicroorganisms and/or are the site of mutations by which suchmicroorganisms acquire drug resistance (e.g., the HIV reversetranscriptase gene). Other reference sequences of interest includeregions where polymorphic variations are known to occur (e.g., theD-loop region of mitochondrial DNA). These reference sequences haveutility for, e.g., forensic or epidemiological studies. Other referencesequences of interest include p34 (related to p53), p65 (implicated inbreast, prostate and liver cancer), and DNA segments encodingcytochromes P450 and other biotransformation genes (see Meyer et al.,Pharmac. Ther. 46, 349-355 (1990)). Other reference sequences ofinterest include those from the genome of pathogenic viruses (e.g.,hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II,and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses,echovirus, rhinovirus, coxsackie virus, cornovirus, respiratorysyncytial virus, mumps virus, rotavirus, measles virus, rubella virus,parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus,molluscum virus, poliovirus, rabies virus, JC virus and arboviralencephalitis virus. Other reference sequences of interest are fromgenomes or episomes of pathogenic bacteria, particularly regions thatconfer drug resistance or allow phylogenic characterization of the host(e.g., 16S rRNA or corresponding DNA). For example, such bacteriainclude chlamydia, rickettsial bacteria, mycobacteria, staphylococci,treptocci, pneumonococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, legionella, diphtheria, salmonella,bacilli, cholera, tetanus, botulism, anthrax, plague, leptospiros is,and Lymes disease bacteria. Other reference sequences of interestinclude those in which mutations result in the following autosomalrecessive disorders: sickle cell anemia, β-thalassemia, phenylketonuria,galactosemia, Wilson's disease, hemochromatosis, severe combinedimmunodeficiency, alpha-1-antitrypsin deficiency, albinism,alkaptonuria, lysosomal storage diseases and Ehlers-Danlos syndrome.Other reference sequences of interest include those in which mutationsresult in X-linked recessive disorders: hemophilia, glucose-6-phosphatedehydrogenase, agammaglobulimenia, diabetes insipidus, Lesch-Nyhansyndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's diseaseand fragile X-syndrome. Other reference sequences of interest includesthose in which mutations result in the following autosomal dominantdisorders: familial hypercholesterolemia, polycystic kidney disease,Huntingdon's disease, hereditary spherocytosis, Marfan's syndrome, vonWillebrand's disease, neurofibromatosis, tuberous sclerosis, hereditaryhemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlossyndrome, myotonic dystrophy, muscular dystrophy, osteogenesisimperfecta, acute intermittent porphyria, and von Hippel-Lindau disease.

The length of a reference sequence can vary widely from a full-lengthgenome, to an individual chromosome, episome, gene, component of a gene,such as an exon, intron or regulatory sequences, to a few nucleotides. Areference sequence of between about 2, 5, 10, 20, 50, 100, 5000, 1000,5,000 or 10,000, 20,000 or 100,000 nucleotides is common. Sometimes onlyparticular regions of a sequence (e.g., exons of a gene) are ofinterest. In such situations, the particular regions can be consideredas separate reference sequences or can be considered as components of asingle reference sequence, as matter of arbitrary choice.

A reference sequence can be any naturally occurring, mutant, consensusor purely hypothetical sequence of nucleotides, RNA or DNA. For example,sequences can be obtained from computer data bases, publications or canbe determined or conceived de novo. Usually, a reference sequence isselected to show a high degree of sequence identity to envisaged targetsequences. Often, particularly, where a significant degree of divergenceis anticipated between target sequences, more than one referencesequence is selected. Combinations of wildtype and mutant referencesequences are employed in several applications of the tiling strategy.

B. Chin Design

1. Basic Tiling Strategy

The basic tiling strategy provides an array of immobilized probes foranalysis of target sequences showing a high degree of sequence identityto one or more selected reference sequences. The strategy is firstillustrated for an array that is subdivided into four probe sets,although it will be apparent that in some situations, satisfactoryresults are obtained from only two probe sets. A first probe setcomprises a plurality of probes exhibiting perfect complementarity witha selected reference sequence. The perfect complementarity usuallyexists throughout the length of the probe. However, probes having asegment or segments of perfect complementarily that is/are flanked byleading or trailing sequences lacking complementarity to the referencesequence can also be used. Within a segment of complementarity, eachprobe in the first probe set has at least one interrogation positionthat corresponds to a nucleotide in the reference sequence. That is, theinterrogation position is aligned with the corresponding nucleotide inthe reference sequence, when the probe and reference sequence arealigned to maximize complementarity between the two. If a probe has morethan one interrogation position, each corresponds with a respectivenucleotide in the reference sequence. The identity of an interrogationposition and corresponding nucleotide in a particular probe in the firstprobe set cannot be determined simply by inspection of the probe in thefirst set. As will become apparent, an interrogation position andcorresponding nucleotide is defined by the comparative structures ofprobes in the first probe set and corresponding probes from additionalprobe sets.

In principle, a probe could have an interrogation position at eachposition in the segment complementary to the reference sequence.Sometimes, interrogation positions provide more accurate data whenlocated away from the ends of a segment of complementarity. Thus,typically a probe having a segment of complementarity of length x doesnot contain more than x-2 interrogation positions. Since probes aretypically 9-21 nucleotides, and usually all of a probe is complementary,a probe typically has 1-19 interrogation positions. Often the probescontain a single interrogation position, at or near the center of probe.

For each probe in the first set, there are, for purposes of the presentillustration, up to three corresponding probes from three additionalprobe sets. See FIG. 1. Thus, there are four probes corresponding toeach nucleotide of interest in the reference sequence. Each of the fourcorresponding probes has an interrogation position aligned with thatnucleotide of interest. Usually, the probes from the three additionalprobe sets are identical to the corresponding probe from the first probeset with one exception. The exception is that at least one (and oftenonly one).interrogation position, which occurs in the same position ineach of the four corresponding probes from the four probe sets, isoccupied by a different nucleotide in the four probe sets. For example,for an A nucleotide in the reference sequence, the corresponding probefrom the first probe set has its interrogation position occupied by a T,and the corresponding probes from the additional three probe sets havetheir respective interrogation positions occupied by A, C, or G, adifferent nucleotide in each probe. Of course, if a probe from the firstprobe set comprises trailing or flanking sequences lackingcomplementarity to the reference sequences (see. FIG. 2), thesesequences need not be present in corresponding probes from the threeadditional sets. Likewise corresponding probes from the three additionalsets can contain leading or trailing sequences outside the segment ofcomplementarity that are not present in the corresponding probe from thefirst probe set. Occasionally, the probes from the additional threeprobe set are identical (with the exception of interrogationposition(s)) to a contiguous subsequence of the full complementarysegment of the corresponding probe from the first probe set. In thiscase, the subsequence includes the interrogation position and usuallydiffers from the full-length probe only in the omission of one or bothterminal nucleotides from the termini of a segment of complementarity.That is, if a probe from the first probe set has a segment ofcomplementarity of length n, corresponding probes from the other setswill usually include a subsequence of the segment of at least lengthn−2. Thus, the subsequence is usually at least 3, 4, 7, 9., 15, 21, or25 nucleotides long, most typically, in the range of 9-21 nucleotides.The subsequence should be sufficiently long to allow a probe tohybridize detectably more strongly to a variant of the referencesequence mutated at the interrogation position than to the referencesequence.

The probes can be oligodeoxyribonucleotides or oligoribonucleotides, orany modified forms of these polymers that are capable of hybridizingwith a target nucleic sequence by complementary base-pairing.Complementary base pairing means sequence-specific base pairing whichincludes e.g., Watson-Crick base pairing as well as other forms of basepairing such as Hoogsteen base pairing. Modified forms include2′-O-methyl oligoribonucleotides and so-called. PNAs, in whicholigodeoxyribonucleotides are linked via peptide bonds rather thanphophodiester bonds. The probes can be attached by any linkage to asupport (e.g., 3′, 5′ or via the base). 3′ attachment is more usual asthis orientation is compatible with the preferred chemistry for solidphase synthesis of oligonucleotides.

The number of probes in the first probe set (and as a consequence thenumber of probes in additional probe sets) depends on the length of thereference sequence, the number of nucleotides of interest in thereference sequence and the number of interrogation positions per probe.In general, each nucleotide of interest in the reference sequencerequires the same interrogation position in the four sets of probes.Consider, as an example, a reference sequence of 100 nucleotides, 50 ofwhich are of interest, and probes each having a single interrogationposition. In this situation, the first probe set requires fifty probes,each having one interrogation position corresponding to a nucleotide ofinterest in the reference sequence. The second, third and fourth probesets each have a corresponding probe for each probe in the first probeset, and so each also contains a total of fifty probes. The identity ofeach nucleotide of interest in the reference sequence is determined bycomparing the relative hybridization signals at four probes havinginterrogation positions corresponding to that nucleotide from the fourprobe sets.

In some reference sequences, every nucleotide is of interest. In otherreference sequences, only certain portions in which variants (e.g.,mutations or polymorphisms) are concentrated are of interest. In otherreference sequences, only particular mutations or polymorphisms andimmediately adjacent nucleotides are of interest. Usually, the firstprobe set has interrogation positions selected to correspond to at leasta nucleotide (e.g., representing a point mutation) and one immediatelyadjacent nucleotide. Usually, the probes in the first set haveinterrogation positions corresponding to at least 3, 10, 50, 100, 1000,or 20,000 contiguous nucleotides. The probes usually have interrogationpositions corresponding to at least 5, 10, 30, 50, 75, 90, 99 orsometimes 100% of the nucleotides in a reference sequence. Frequently,the probes in the first probe set completely span the reference sequenceand overlap with one another relative to the reference sequence. Forexample, in one common arrangement each probe in the first probe setdiffers from another probe in that set by the omission of a 3′ basecomplementary to the reference sequence and the acquisition of a 5′ basecomplementary to the reference sequence. See FIG. 3A.

The number of probes on the chip can be quite large (e.g., 10⁵-10⁶).However, often only a relatively small proportion (i.e., less than about50%, 25%, 10%, 5% or 1%) of the total number of probes of a given lengthare selected to pursue a particular tiling strategy. For example, acomplete set of octomer probes comprises 65,536 probes; thus, an arrayof the invention typically has fewer than 32,768 octomer probes. Acomplete array of decamer probes comprises 1,048,576 probes; thus, anarray of the invention typically has fewer than about-500,000 decamerprobes. Often arrays have a lower limit of 25, 50 or 100 probes and anupper limit of 1,000,000, 100,,000, 10,000 or 1000 probes. The arrayscan have other components besides the probes such as linkets attachingthe probes to a support.

Some advantages of the use of only a proportion of all possible probesof a given length include: (i) each position in the array is highlyinformative, whether or not hybridization occurs; (ii) nonspecifichybridization is minimized; (iii) it is straightforward to correlatehybridization differences with sequence differences, particularly withreference to the hybridization pattern of a known standard; and (iv) theability to address each probe independently during synthesis, using highresolution photolithography, allows the array to be designed andoptimized for any sequence. For example the length of any probe can bevaried independently of the others.

For conceptual simplicity, the probes in a set are usually arranged inorder of the sequence in a lane across the chip. A lane contains aseries of overlapping probes, which represent or tile across, theselected reference sequence (see FIG. 3A). The components of the foursets of probes are usually laid down in four parallel lanes,collectively constituting a row in the horizontal direction and a seriesof 4-member columns in the vertical direction. Corresponding probes fromthe four probe sets (i.e., complementary to the same subsequence of thereference sequence) occupy a column. Each probe in a lane usuallydiffers from its predecessor in the lane by the omission of a base atone end and the inclusion of additional base at the other end as shownin FIG. 3A. However, this orderly progression of probes can beinterrupted by the inclusion of control probes or omission of probes incertain columns of the array. Such columns serve as controls to orientthe chip, or gauge the background, which can include target sequencenonspecifically bound to the chip.

The probes sets are usually laid down in lanes such that all probeshaving an interrogation position occupied by an A form an A-lane, allprobes having an interrogation position occupied by a C form a C-lane,all probes having an interrogation position occupied by a G form aG-lane, and all probes having an interrogation position occupied by a T(or U) form a T lane (or a U lane). Note that in this arrangement thereis not a unique correspondence between probe sets and lanes. Thus, theprobe from the first probe set is laid down in the A-lane, C-lane,A-lane, A-lane and T-lane for the five columns in FIG. 4A. Theinterrogation position on a column of probes corresponds to the positionin the target sequence whose identity is determined from analysis ofhybridization to the probes in that column. Thus, I₁-I₅ respectivelycorrespond to N₁-N₅ in FIG. 4A. The interrogation position an beanywhere in a probe but is usually at or near the central position ofthe probe to maximize differential hybridization signals between aperfect match and a single-base mismatch. For example, for an 11 merprobe, the central position is the sixth nucleotide.

Although the array of probes is usually laid down in rows and columns asdescribed above, such a physical arrangement of probes on the chip isnot essential. Provided that the spatial location of each probe in anarray is known, the data from the probes can be collected and processedto yield the sequence of a target irrespective of the physicalarrangement of the probes on a chip. In processing the data, thehybridization signals from the respective probes can be reasserted intoany conceptual array desired for subsequent data reduction whatever thephysical arrangement of probes on the chip.

A range of lengths of probes can be employed in the chips. As notedabove, a probe may consist exclusively of a complementary segments, ormay have one or more complementary segments juxtaposed by flanking,trailing and/or intervening segments. In the latter situation, the totallength of complementary segment(s) is more important that the length ofthe probe. In functional terms, the complementary segment(s) -of thefirst probe sets should be sufficiently long to allow the probe tohybridize detectably more strongly to a reference sequence compared witha variant of the reference including a single base mutation at thenucleotide corresponding to the interrogation position of the probe.Similarly, the complementary segment(s) in corresponding probes fromadditional probe sets should be sufficiently long to allow a probe tohybridize detectably more strongly to a variant of the referencesequence having a single nucleotide substitution at the interrogationposition relative to the reference sequence. A probe usually has asingle complementary segment having a length of at least 3 nucleotides,and more usually at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or 30 bases exhibiting perfectcomplementarity (other than possibly at the interrogation position(s)depending on the probe set) to the reference sequence. In bridgingstrategies, where more than one segment of complementarity is present,each segment provides at least three complementary nucleotides to thereference sequence and the combined segments provide at least twosegments of three or a total of six complementary nucleotides. As in theother strategies, the combined length of complementary segments istypically from 6-30 nucleotides, and preferably from about 9-21nucleotides. The two segments are often approximately the same length.Often, the probes (or segment of complementarity within probes) have anodd number of bases, so that an interrogation position can occur in theexact center of the probe.

In some chips, all probes are the same length.

Other chips employ different groups of probe sets, in which case theprobes are of the same size within a group, but differ between differentgroups. For example, some chips have one group comprising four sets ofprobes as described above in which all the probes are 11 mers, togetherwith a second group comprising four sets of probes in which, all of theprobes are 13 mers. Of course, additional groups of probes can be added.

Thus, some chips contain, e.g., four groups of probes having sizes of 11mers, 13 mers, 15 mers and 17 mers. Other chips have different sizeprobes within the same group of four probe sets. In these chips, theprobes in the first set can vary in length independently of each other.Probes in the other sets are usually the same length as the probeoccupying the same column from the first set. However, occasionallydifferent lengths of probes can be included at the same column positionin the four lanes. The different length probes are included equalizehybridization signals from probes irrespective of whether A-T or C-Gbonds are formed at the interrogation position.

The length of probe can be important in distinguishing between aperfectly matched probe and probes showing a single base mismatch withthe target sequence. The discrimination is usually greater for shortprobes. Shorter probes are usually also less susceptible to formation ofsecondary structures. However, the absolute amount of target sequencebound, and hence the signal, is greater for larger probes. The probelength representing the optimum compromise between these competingconsiderations may vary depending on inter alia the GC content of aparticular region of the target DNA sequence, secondary structure,synthesis efficiency and cross-hybridization. In some regions of thetarget, depending on hybridization conditions, short probes (e.g., 11mers) may provide information that is inaccessible from longer probes(e.g., 19 mers) and vice versa. Maximum sequence information can be readby including several groups of different sized probes on the chip asnoted above. However, for many regions of the target sequence, such astrategy provides redundant information in that the same sequence isread multiple times from the different groups of probes. Equivalentinformation can be obtained from a single group of different sizedprobes in which the sizes are selected to maximize readable sequence Sat particular regions of the target sequence. The strategy ofcustomizing probe length within a single group of probe sets minimizesthe total number of probes required to read a particular targetsequence. This leaves ample capacity for the chip to include probes toother reference sequences.

The invention provides an optimization block which allows systematicvariation of probe length and interrogation position to optimize theselection of probes for analyzing a particular nucleotide in a referencesequence. The block comprises-alternating columns of probescomplementary to the wildtype target and probes complementary to aspecific mutation. The interrogation position is varied between columnsand probe length is varied down a column.

Hybridization of the chip to the reference sequence or the mutant formof the reference sequence identifies the probe length and interrogationposition providing the greatest differential hybridization signal.

Variation of interrogation position in probes for analyzing differentregions of a target sequence offers a number of advantages. If a segmentof a target sequence contains two closely spaced mutations, m1, and m2,and probes for analyzing that segment have an interrogation position ator near the middle, then no probe has an interrogation position alignedwith one of the mutations without overlapping the other mutation (seefirst probe in FIG. 4B). Thus, the presence of a mutation would have tobe detected by comparing the hybridization signal of a single-mismatchedprobe with a double-mismatched probe. By contrast, if the interrogationposition is near the 3′ end of the probes, probes can have theirinterrogation position aligned with m1 without overlapping m2 (secondprobe in FIG. 4B). Thus, the mutation can be detected by a comparison ofa perfectly matched probe with single based mismatched probes.Similarly, if the interrogation position is near the 5′ end of theprobes, probes can have their interrogation position aligned with m2without overlapping m1 (third probe in FIG. 4B).

Variation of the interrogation position also offers the advantage ofreducing loss of signal due to self-annealing of certain probes. FIG. 4Cshows a target sequence having a nucleotide X, which can be read eitherfrom the relative signals of the four probes having a centralinterrogation position (shown at the left of the figure) or from thefour probes having the interrogation position near the three prime end(shown at the right of the figure). Only the probes having the centralinterrogation position are capable of self-annealing. Thus, a highersignal is obtained from the probes having the interrogation positionnear the terminus.

The probes are designed to be complementary to either strand of thereference sequence (e.g., coding or non-coding). Some chips containseparate groups of probes, one complementary to the coding strand, theother complementary to the noncoding strand. Independent analysis ofcoding and noncoding strands provides largely redundant information.However, the regions of ambiguity in reading the coding strand are notalways the same as those in reading the noncoding strand. Thus,combination of the information from coding and noncoding strandsincreases the overall accuracy of sequencing.

Some chips contain additional probes or groups of probes designed to becomplementary to a second reference sequence. The second referencesequence is often a subsequence of the first reference sequence bearingone or more commonly occurring mutations or interstrain variations. Thesecond group of probes is designed by the same principles as describedabove except that the probes exhibit complementarity to the secondreference sequence. The inclusion of a second group is particular usefulfor analyzing short subsequences of the primary reference sequence inwhich multiple mutations are expected to occur within a short distancecommensurate with the length of the probes (i.e., two or more mutationswithin 9 to 21 bases). Of course, the same principle can be extended toprovide chips containing groups of probes for any number of referencesequences. Alternatively, the chips may contain additional probe(s) thatdo not form part of a tiled array as noted above, but rather serves asprobe(s) for a conventional reverse dot blot. For example, the presenceof mutation can be detected from binding of a target sequence to asingle oligomeric probe harboring the mutation. Preferably, anadditional probe containing the equivalent region of the wildtypesequence is included as a control.

Although only a subset of probes is required to analyze a particulartarget sequence, it is quite possible that other probes superfluous tothe contemplated analysis are also included on the chip. In the extremecase, the chip could can a complete set of all probes of a given lengthnotwithstanding that only a small subset is required to analyze theparticular reference sequence of interest. Although such a situationmight appear wasteful of resources, a chip including a complete set ofprobes offers the advantage of including the appropriate subset ofprobes for analyzing any reference sequence. Such a chip also allowssimultaneous analysis of a reference sequence from different subsets ofprobes (e.g., subsets having the interrogation site at differentpositions in the probe).

In its simplest terms, the analysis of a chip reveals whether the targetsequence is the same or different from the reference sequence. If thetwo are the same, all probes in the first probe set show a strongerhybridization signal than corresponding probes from other probe sets. Ifthe two are different, most probes from the first probe set still show astronger hybridization signal than corresponding probes from the otherprobe sets, but some probes from the first probe set do not. Thus, whena probe from another probe sets light up more strongly than thecorresponding probe from the first probe set, this provides a simplevisual indication that the target sequence and reference sequencediffer.

The chips also reveal the nature and position of differences between thetarget and reference sequence. The chips are read by comparing theintensities of labelled target bound to the probes in an array.Specifically, for each nucleotide of interest in the target sequence, acomparison is performed between probes having an interrogation positionaligned with that position. These probes form a column (actual orconceptual) on the chip. For example, a column often contains one probefrom each of A, C, G and T lanes. The nucleotide in the target sequenceis identified as the complement of the nucleotide occupying theinterrogation position in the probe showing the highest hybridizationsignal from a column. FIG. 6 shows the hybridization pattern of a chiphybridized to its reference sequence. The dark square in each columnrepresents the probe from the column having the highest hybridizationsignal. The sequence can be read by following the pattern of darksquares from left to right across the chip. The first dark square is inthe A lane indicating that the nucleotide occupying the interrogationposition of the probe represented by this square is an A. The firstnucleotide in the reference sequence is the complement of nucleotideoccupying the interrogation position of this probe (i.e., a T).Similarly, the second dark square is in the T-lane, from which it can bededuced that the second nucleotide in the reference sequence is an A.Likewise the third dark square is in the T-lane, from which it can bededuced that the third nucleotide in the reference sequence is also anA, and so forth. By including probes in the first probe set (and byimplication in the other probe sets) with interrogation positionscorresponding to every nucleotide in a reference sequence, it ispossible to read substantially every nucleotide in a target sequence,thereby revealing the complete or nearly complete sequence of thetarget.

Of the four probes in a column, only one can exhibit a perfect match tothe target sequence whereas the others usually exhibit at least a onebase pair mismatch. The probe exhibiting a perfect match usuallyproduces a substantially greater hybridization signal than the otherthree probes in the column and is thereby easily identified. However, insome regions of the target sequence, the distinction between a perfectmatch and a one-base mismatch is less clear. Thus, a call ratio isestablished to define the ratio of signal from the best hybridizingprobes to the second best hybridizing probe that must be exceeded for aparticular target position to be read from the probes. A high call ratioensures that few if any errors are made in calling target nucleotides,but can result in some nucleotides being scored as ambiguous, whichcould in fact be accurately read. A lower call ratio results in fewerambiguous calls, but can result in more erroneous calls. It has beenfound that at a call ratio of 1.2 virtually all calls are accurate.However, a small but significant number of bases (e.g., up to about 10%)may have to be scored as ambiguous.

Although small regions of the target sequence can sometimes beambiguous, these regions usually occur at the same or similar segmentsin different target sequences. Thus, for precharacterized mutations, itis known in advance whether that mutation is likely to occur within aregion of unambiguously determinable sequence.

An array of probes is most useful for analyzing the reference sequencefrom which the probes were designed and variants of that sequenceexhibiting substantial sequence similarity with the reference sequence(e.g., several single-base mutants spaced over the reference sequence).When an array is used to analyze the exact reference sequence from whichit was designed, one probe exhibits a perfect match to the referencesequence, and the other three probes in the same column exhibitssingle-base mismatches. Thus, discrimination between hybridizationsignals is usually high and accurate sequence is obtained. High accuracyis also obtained when an array is used for analyzing a target sequencecomprising a variant of the reference sequence that has a singlemutation relative to the reference sequence, or several widely spacedmutations relative to the reference sequence. At different mutant loci,one probe exhibits a perfect match to the target, and the other threeprobes occupying the same column exhibit single-base mismatches, thedifference (with respect to analysis of the reference sequence) beingthe lane in which the perfect match occurs.

For target sequences showing a high degree of divergence from thereference strain or incorporating several closely spaced mutations fromthe-reference strain, a single group of probes (i.e., designed withrespect to a single reference sequence) will not always provide accuratesequence for the highly variant region of this sequence. At someparticular columnar positions, it may be that no single probe exhibitsperfect complementarity to the target and that any comparison must bebased on different degrees of mismatch between the four probes. Such acomparison does not always allow the target nucleotide corresponding tothat columnar position to be called. Deletions in target sequences canbe detected by loss of signal from probes having interrogation positionsencompassed by the deletion. However, signal may also be lost fromprobes having interrogation positions closely proximal to the deletionresulting in some regions of the target sequence that cannot be read.Target sequence bearing insertions will also exhibit short regionsincluding and proximal to the insertion that usually cannot be read.

The presence of short regions of difficult-to-read target because ofclosely spaced mutations, insertions or deletions, does not preventdetermination of the remaining sequence of the target as differentregions of a target sequence are determined independently.Moreover,.such ambiguities as might result from analysis of diversevariants with a single group of probes can be avoided by includingmultiple groups of probe sets on a chip. For example, one group ofprobes can be designed based on a full-length reference sequence, andthe other groups on subsequences of the reference sequence incorporatingfrequently occurring mutations or strain variations.

A particular advantage of the present sequencing strategy overconventional sequencing methods is the capacity simultaneously to detectand quantify proportions of multiple target sequences. Such capacity isvaluable, e.g., for diagnosis of patients who are heterozygous withrespect to a gene or who are infected with a virus, such as HIV, whichis usually present in several polymorphic forms. Such capacity is alsouseful in analyzing targets from biopsies of tumor cells and surroundingtissues. The presence of multiple target sequences is detected from therelative signals of the four probes at the array columns correspondingto the target nucleotides at which diversity occurs. The relativesignals of the four probes for the mixture under test are compared withthe corresponding signals from a homogeneous reference sequence. Anincrease in a signal from a probe that is mismatched with respect to thereference sequence, and a corresponding decrease in the signal from theprobe which is matched with the reference sequence, signal the presenceof a mutant strain in the mixture. The extent in shift in hybridizationsignals of the probes is related to the proportion of a target sequencein the mixture. Shifts in relative hybridization signals can bequantitatively related to proportions of reference and mutant sequenceby prior calibration of the chip with seeded mixtures of the mutant andreference sequences. By this means, a chip can be used to detect variantor mutant strains constituting as little as 1, 5, 20, or 25% of amixture of stains.

Similar principles allow the simultaneous analysis of multiple targetsequences even when none is identical to the reference sequence. Forexample, with a mixture of two target sequences bearing first and secondmutations, there would be a variation in the hybridization patterns ofprobes having interrogation positions corresponding to the first andsecond mutations relative to the hybridization pattern with thereference sequence. At each position, one of the probes having amismatched interrogation position relative to the reference sequencewould show an increase in hybridization signal, and the probe having amatched interrogation position relative to the reference sequence wouldshow a decrease in hybridization signal. Analysis of the hybridizationpattern of the mixture of mutant target sequences, preferably incomparison with the hybridization pattern of the reference sequence,indicates the presence of two mutant target sequences, the position andnature of the mutation in each strain, and the relative proportions ofeach strain.

In a variation of the above method, several target sequences targetsequences are differentially labelled before being simultaneouslyapplied to the array. For example, each different target sequence can belabelled with a fluorescent labels emitting at different wavelength.After applying a mixtures of target sequence to the arrays, theindividual target sequences can be distinguished and independentlyanalyzed by virtue of the differential labels. For example, the methodstarget sequences obtained from a patient at different stages of adisease can be differently labelled and analyzed simultaneously,facilitating identification of new mutations.

2. Omission of Probes

The basic strategy outlined above employs four probes to read eachnucleotide of interest in a target sequence. One probe (from the firstprobe set) shows a perfect match to the reference sequence and the otherthree probes (from the second, third and fourth probe sets) exhibit amismatch with the reference sequence and a perfect match with a targetsequence bearing a mutation at the nucleotide of interest. The provisionof three probes from the second, third and fourth probe sets allowsdetection of each of the three possible nucleotide substitutions of anynucleotide of interest. However, in some reference -sequences or regionsof reference sequences, it is known in advance that only certainmutations are likely to occur. Thus, for example, at one site it mightbe known that an A nucleotide in the reference sequence may exist as a Tmutant in some target sequences but s unlikely to exist as a C or Gmutant. Accordingly, for analysis of this region of the referencesequence, one might include only the first and second probe sets, thefirst probe set exhibiting perfect complementarity to the referencesequence, and the second probe set having an interrogation positionoccupied by an invariant A residue (for detecting the T mutant). Inother situations, one might include the first, second and third probessets (but not the fourth) for detection of a wildtype nucleotide in thereference sequence and two mutant variants thereof in target sequences.In some chips, probes that would detect silent mutations (i.e., notaffecting amino acid sequence) are omitted.

Some chips effectively contain the second, third and optionally, thefourth probes sets described in the basic tiling strategy (i.e., themismatched probe sets) but omit some or all of the probes from the firstprobe set (i.e., perfectly matched probes). Therefore, such chipscomprise at least two probe sets, which will arbitrarily be referred toas probe sets A and B (to avoid confusion with the nomenclature used todescribe the four probe sets in the basic tiling strategy). Probe set Ahas a plurality of probes. Each probe comprises a segment exactlycomplementary to a subsequence of a reference sequence except in atleast one interrogation position. The interrogation position correspondsto a nucleotide in the reference sequence juxtaposed with theinterrogation position when the reference sequence and probe aremaximally aligned. Probe set B has a corresponding probe for each probein the first probe set. The corresponding probe in probe set B isidentical to a sequence comprising the corresponding probe from thefirst probe set or a subsequence thereof that includes the at least one(and usually only one) interrogation position except that the at leastone interrogation position is occupied by a different nucleotide in eachof the two corresponding probes from the probe sets A and B. Anadditional probe set C, if present, also comprises a corresponding probefor each probe in the probe set A except in the at least oneinterrogation position, which differs in the corresponding probes fromprobe sets A, B and C. The arrangement of probe sets A, B and C is shownin FIG. 3B. FIG. 3B is the same as FIG. 3A except that the first probeset has been omitted and the second, third and fourth probe sets in FIG.3A have been relabelled as probe sets A, B and C in FIG. 3B.

Chips lacking perfectly matched probes are preferably analyzed byhybridization to both target and reference sequences. The hybridizationscan be performed sequentially, or, if the target and reference aredifferentially labelled, concurrently. The hybridization data are thenanalyzed in two ways. First, considering only the hybridization signalsof the probes to the target sequence, one compares the signals ofcorresponding probes for each position of interest in the targetsequence. For a position of mismatch with the reference sequence, one ofthe probes having an interrogation position aligned with that positionin the target sequence shows a substantially higher signal than othercorresponding probes. The nucleotide occupying the position of mismatchin the target sequence is the complement of the nucleotide occupying theinterrogation position of the corresponding probe showing the highestsignal. For a position where target and reference sequence are the same,none of the corresponding probes having an interrogation positionaligned with that position in the target sequence is matched, andcorresponding probes generally show weak signals, which may varysomewhat from each other.

In a second level of analysis, the ratio of hybridization signals to thetarget and reference sequences is determined for each probe in thearray. For most probes in the array the ratio of hybridization signalsis about the same. For such a probe, it can be deduced that theinterrogation position of the probe corresponds to a nucleotide that isthe same in target and reference sequences. A few probes show a muchhigher ratio of target hybridization to reference hybridization than themajority of probes. For such a probe, it can be deduced that theinterrogation position of the probe corresponds to a nucleotide thatdiffers between target and reference sequences, and that in the target,this nucleotide is the complement of the nucleotide occupying theinterrogation position of the probe. The second level of analysis servesas a control to confirm the identification of differences-between targetand reference sequence from the first level of analysis.

3. Wildtype Probe Lane

When the chips comprise four probe sets, as discussed supra, and theprobe sets are laid down in four lanes, an A lane, a C-lane, a G laneand a T or U lane, the probe having a segment exhibiting perfectcomplementarity to a reference sequence varies between the four lanesfrom one column to another. This does not present any significantdifficulty in computer analysis of the data from the chip. However,visual inspection of the hybridization pattern of the chip is sometimesfacilitated by provision of an extra lane of probes, in which each probehas a segment exhibiting perfect complementarity to the referencesequence. See FIG. 4A. This extra lane of probes is called the wildtypelane and contains only probes from the first probe set. Each wildtypelane probe has a segment that is identical to a segment from one of theprobes in the other four lanes (which lane depending on the columnposition). The wildtype lane hybridizes to a target sequence at allnucleotide positions except those in which deviations from the referencesequence occurs. The hybridization pattern of the wildtype lane therebyprovides a simple visual indication of mutations.

4. Deletion, Insertion and Multiple-Mutation Probes

Some chips provide an additional probe set specifically designed foranalyzing deletion mutations. The additional probe set comprises a probecorresponding to each probe in the first probe set as described above.However, a probe from the additional probe set differs from thecorresponding probe in the-first probe set in that the nucleotideoccupying the interrogation position is deleted in the probe from theadditional probe set. See FIG. 6. Optionally, the probe from theadditional probe set bears an additional nucleotide at one of itstermini relative to the corresponding probe from the first probe set(shown in brackets in FIG. 6). The probe from the additional probe setwill hybridize more strongly than the corresponding probe from the firstprobe set to a target sequence having a single base deletion at thenucleotide corresponding to the interrogation position. Additional probesets are provided in which not only the interrogation position, but alsoan adjacent nucleotide is deleted.

Similarly, other chips provide additional probe sets for analyzinginsertions. For example, one additional probe set has a probecorresponding to each probe in the first probe set as described above.However, the probe in the additional probe set has an extra T nucleotideinserted adjacent to the interrogation position. See FIG. 6 (the extra Tis shown in a square box). Optionally, the probe has one fewernucleotide at one of its termini relative to the corresponding probefrom the first probe set (shown in brackets). The probe from theadditional probe set hybridizes more strongly than the correspondingprobe from the first probe set to a target sequence having an Ainsertion to the left of nucleotide “n” the reference sequence in FIG.6. Similar additional probe sets can be constructed having C, G or Anucleotides inserted adjacent to the interrogation position.

Usually, four such additional probe sets, one for each nucleotide, areused in combination. Comparison of the hybridization signal of theprobes from the additional probe sets with the corresponding probe fromthe first probe set indicates whether the target sequence contains andinsertion. For example, if a probe from one of the additional probe setsshows a higher hybridization signal than a corresponding probe From thefirst probe set, it is deduced that the target sequence contains aninsertion adjacent to the corresponding nucleotide (n) in the targetsequence. The inserted base in the target is the complement of theinserted base in the probe from the additional probe set showing thehighest hybridization signal. If the corresponding probe from the firstprobe set shows a higher hybridization signal than the correspondingprobes from the additional probe sets, then the target sequence does notcontain an insertion to the left of corresponding position ((“n” in FIG.6)) in the target sequence.

Other chips provide additional probes (multiple-mutation probes) foranalyzing target sequences having multiple closely spaced mutations. Amultiple-mutation probe is usually identical to a corresponding probefrom the first set as described above, except in the base occupying theinterrogation position, and except at one or more additional positions,corresponding to nucleotides in which substitution may occur in thereference sequence. The one or more additional positions in the multiplemutation probe are occupied by nucleotides complementary to thenucleotides occupying corresponding positions in the reference sequencewhen the possible substitutions have occurred.

5. Block Tiling

In block tiling, a perfectly matched (or wildtype) probe is comparedwith multiple sets of mismatched or mutant probes. The perfectly matchedprobe and the multiple sets of mismatched probes with which it iscompared collectively form a group or block of probes on the chip. Eachset comprises at least one, and usually, three mismatched probes. FIG. 7shows a perfectly matched probe (CAATCGA) having three interrogationpositions (I₁, I₂ and I₃). The perfectly matched probe is compared withthree sets of probes (arbitrarily designated A, B and C), each havingthree mismatched probes. In set A, the three mismatched probes areidentical to a sequence comprising the perfectly matched probe or asubsequence thereof including the interrogation positions, except at thefirst interrogation position. That is, the mismatched probes in the setA differ from the perfectly matched probe set at the first interrogationposition. Thus, the relative hybridization signals of the perfectlymatched probe and the mismatched probes in the set A indicates theidentity of the nucleotide in a target sequence corresponding to thefirst interrogation position. This nucleotide is the complement of thenucleotide occupying the interrogation position of the probe showing thehighest signal. Similarly, set B comprises three mismatched probes, thatdiffer from the perfectly matched probe at the second interrogationposition. The relative hybridization intensities of the perfectlymatched probe and the three mismatched probes of set B reveal theidentity of the nucleotide in the target sequence corresponding to thesecond interrogation position (i.e., n2, in FIG. 7). Similarly, thethree mismatched probes in set C in FIG. 7 differ from the perfectlymatched probe at the third interrogation position. Comparison of thehybridization intensities of the perfectly matched probe and themismatched probes in the set C reveals the identity of the nucleotide inthe target sequence corresponding to the third interrogation position(n3).

As noted above, a perfectly matched probe may have seven or moreinterrogation positions. If there are seven interrogation positions,there are seven sets of three mismatched probe, each set serving toidentify the nucleotide corresponding to one of the seven interrogationpositions. Similarly, if there are 20 interrogation positions in theperfectly matched probe, then 20 sets of three mismatched probes areemployed. As in other tiling strategies, selected probes can be omittedif it is known in advance that only certain types of mutations arelikely to arise.

Each block of probes allows short regions of a target sequence to beread. For example, for a block of z,999 aving seven interrogationpositions, seven nucleotides in the target sequence can be read. Ofcourse, a chip can contain any number of blocks depending on how manynucleotides of the target are of interest. The hybridization signals foreach block can be analyzed independently of any other block. The blocktiling strategy can also be combined with other tiling strategies, withdifferent parts of the same reference sequence being tiled by differentstrategies.

The block tiling strategy is a species of the basic tiling strategydiscussed above, in which the probe from the first probe set has morethan one interrogation position. The perfectly matched probe in theblock tiling strategy is equivalent to a probe from the first probe setin the basic tiling strategy. The three mismatched probes in set A inblock tiling are equivalent to probes from the second, third and fourthprobe sets in the basic tiling strategy. The three mismatched probes inset B of block tiling are equivalent to probes from additional probesets in basic tiling arbitrarily designated the fifth, sixth and seventhprobe sets. The three mismatched probes in set C of blocking tiling areequivalent to probes from three further probe sets in basic tilingarbitrarily designated the eighth, ninth and tenth probe sets.

The block tiling strategy offers two advantages over a basic strategy inwhich each probe in the first set has a single interrogation position.One advantage is that the same sequence information can be obtained fromfewer probes. A second advantage is that each of the probes constitutinga block (i.e.,.a probe from the first probe set and a correspondingprobe from each of the other probe sets) can have identical 3′ and 5′sequences, with the variation confined to a central segment containingthe interrogation positions. The identity of 3′ sequence betweendifferent probes simplifies the strategy for solid phase synthesis ofthe probes on the chip and results in more uniform deposition of thedifferent probes on the chip, thereby in turn increasing the uniformityof signal to noise ratio for different regions of the chip.

6. Multiplex Tiling

In the block tiling strategy discussed above, the identity of anucleotide in a target or reference sequence is determined by comparisonof hybridization patterns of one probe having a segment showing aperfect match with that of other probes (usually three other probes)showing a single base mismatch. In multiplex tiling, the identity of atleast two nucleotides in a reference or target sequence is determined bycomparison of hybridization signal intensities of four probes, two ofwhich have a segment showing perfect complementarity or a single basemismatch to the reference sequence, and two of which have a segmentshowing perfect complementarity or a double-base mismatch to a segment.The four probes whose hybridization patterns are to be compared eachhave a segment that is exactly complementary to a reference sequenceexcept at two interrogation positions, in which the segment may or maynot be complementary to the reference sequence. The interrogationpositions correspond to the nucleotides in a reference or targetsequence which are determined by the comparison of intensities. Thenucleotides occupying the interrogation positions in the four probes areselected according to the following rule. The first interrogationposition is occupied by a different nucleotide in each of the fourprobes. The second interrogation position is also occupied by adifferent nucleotide in each of the four probes. In two of the fourprobes, designated the first and second probes, the segment is exactlycomplementary to the reference sequence except at not more than one ofthe two interrogation positions. In other words, one of theinterrogation positions is occupied by a nucleotide that iscomplementary to the corresponding nucleotide from the referencesequence and the other interrogation position may or may not be sooccupied. In the other two of the four probes, designated the third andfourth probes, the segment is exactly complementary to the referencesequence except that both interrogation positions are occupied bynucleotides which are noncomplementary to the respective correspondingnucleotides in the reference sequence.

There are number of ways of satisfying these conditions depending onwhether the two nucleotides in the reference sequence corresponding tothe two interrogation positions are the same or different. If these twonucleotides are different in the reference sequence (probability ¾), theconditions are satisfied by each of the two interrogation positionsbeing occupied by the same nucleotide in any given probe. For example,in the first probe, the two interrogation positions would both be A, inthe second probe, both would be C, in the third probe, each would be G,and in the fourth probe each would be T or U. If the two nucleotides inthe reference sequence corresponding to the two interrogation positionsare different, the conditions noted above are satisfied by each of theinterrogation positions in any one of the four probes being occupied bycomplementary nucleotides. For example, in the first probe, theinterrogation positions could be occupied by A and T, in the secondprobe by C and G, in the third probe by G and C, and in the four probe,by T and A. See (FIG. 8).

When the four probes are hybridized to a target that is the same as thereference sequence or differs from the reference sequence at:one (butnot both) of the interrogation positions, two of the four probes show adouble-mismatch with the target and two probes show a single mismatch.The identity of probes showing these different degrees of mismatch canbe determined from the different hybridization signals. From theidentity of the probes showing the different degrees of mismatch, thenucleotides occupying both of the interrogation positions in the targetsequence can be deduced.

For ease of illustration, the multiplex strategy has been initiallydescribed for the situation where there are two nucleotides of interestin a reference sequence and only four probes in an array. Of course, thestrategy can be extended to analyze any number of nucleotides in atarget sequence by using additional probes. In one variation, each pairof interrogation positions is read from a unique group of four probes.In a block variation, different groups of four probes exhibit the samesegment of complementarity with the reference sequence, but theinterrogation positions move within a block. The block and standardmultiplex tiling variants can of course be used in combination fordifferent regions of a reference sequence. Either or both variants canalso be used in combination with any of the other tiling strategiesdescribed.

7. Helper Mutations

Occasionally, small regions of a reference sequence give a lowhybridization signal as a result of annealing of probes. Theself-annealing reduces the amount of probe effectively available forhybridizing to the target. Although such regions of the target aregenerally small and the reduction of hybridization signal is usually notso substantial as to obscure the sequence of this region, this concerncan be avoided by the use of probes incorporating helper mutations. Ahelper mutation refers to a position of mismatch in a probe other thanat an interrogation position. The helper mutation(s) serve to break-upregions of internal complementarity within a probe and thereby preventannealing. Usually, one or two helper mutations are quite sufficient forthis purpose. The inclusion of helper mutations can be beneficial in anyof the tiling strategies noted above. In general each probe having aparticular interrogation position has the same helper mutation(s). Thus,such probes have a segment in common which shows perfect complementaritywith a reference sequence, except that the segment contains at least onehelper mutation (the same in each of the probes) and at least oneinterrogation position (different in all of the probes). For example, inthe basic tiling strategy, a probe from the first probe set comprises asegment containing an interrogation position and showing..perfectcomplementarity with a reference sequence except for one or two helpermutations. The corresponding probes from the second, third and fourthprobe sets usually comprise the same segment (or sometimes a subsequencethereof including the helper mutation(s) and interrogation position),except that the base occupying the interrogation position varies in eachprobe. See FIG. 9.

Usually, the helper mutation tiling strategy is used in conjunction withone of the tiling strategies described above. The probes containinghelper mutations are used to tile regions of a reference sequenceotherwise giving low hybridization signal.(e.g.,.because ofself-complementarity), and the alternative tiling strategy is used totile intervening regions.

8. Pooling Strategies

Pooling strategies also employ arrays of immobilized probes. Probes areimmobilized in cells of an array, and the hybridization signal of eachcell can be determined independently of any other cell. A particularcell may be occupied by pooled mixture of probes. Although the identityof each probe in the mixture is known, the individual probes in the poolare not separately addressable. Thus, the hybridization signal from acell is the aggregate of that of the different probes occupying thecell. In general, a cell is scored as hybridizing to a target-sequenceif at least one probe occupying the cell comprises a segment exhibitingperfect complementarity to the target sequence..

A simple strategy to show the increased power of pooled strategies overa standard tiling is to create three cells each containing a pooledprobe having a single pooled position, the pooled position being thesame in each of the pooled probes. At the pooled position, there are twopossible nucleotide, allowing the pooled probe to hybridize to twotarget sequences. In tiling terminology, the pooled position of eachprobe is an interrogation position. As will become apparent, comparisonof the hybridization intensities of the pooled probes from the threecells reveals the identity of the nucleotide in the target sequencecorresponding to the interrogation position (i.e., that is matched withthe interrogation position when the targets sequence and pooled probesare maximally aligned for complementarity)

The three cells are assigned probe pools that are perfectlycomplementary to the target except at the pooled position, which isoccupied by a different pooled nucleotide in each probe as follows:

[AC]=M, [GT]=K, [AG]=R as substitutions in the probe IUPAC standardambiguity notation) X - interrogation position Target:TAACCACTCACGGGAGCA Pool 1: ATTGGMGAGTGCCC =ATTGGaGAGTGCCC (complement tomutant ‘t’) +ATTGGcGAGTGCCC (complement to mutant ‘g’) Pool 2:ATTGGKGAGTGCCC =ATTGGgGAGTGCCC (complement to mutant ‘c’)+ATTGGtGAGTGCCC (complement to wild type ‘a’) Pool 3: ATTGGRGAGTGCCC=ATTGGaGAGTGCCC (complement to mutant ‘t’) +ATTGGgGAGTGCCC (complementto mutant ‘c’)

With 3 pooled probes, all 4 possible single base pair states (wild and 3mutants) are detected. A pool hybridizes with a target if some probecontained within that pool is complementary to that target.Hybridization? Pool: 1 2 3 Target: TAACCACTCACGGGAGCA n y n Mutant:TAACCcCTCACGGGAGCA n y y Mutant: TAACCgCTCACGGGAGCA y n n Mutant:TAACCtCTCACGGGAGCA y n y

A cell containing a pair (or more) of oligonucleotides lights up when atarget complementary to any of the oligonucleotide in the cell ispresent. Using the simple strategy, each of the four possible targets(wild and three mutants) yields a unique hybridization pattern among thethree cells.

Since a different pattern of hybridizing pools is obtained for eachpossible nucleotide in the target sequence corresponding to the pooledinterrogation position in the probes, the identity of the nucleotide canbe determined from the hybridization pattern of the pools. Whereas, astandard tiling requires four cells to detect and identify the possiblesingle-base substitutions at one location, this simple pooled strategyonly requires three cells.

A more efficient pooling strategy for sequence analysis is the ‘Trellis’strategy. In this strategy, each pooled probe has a segment of perfectcomplementarity to a reference sequence except at three pooledpositions. One pooled position is an N pool (IUPAC standard ambiguitycode). The three pooled positions may or may not be contiguous in aprobe. The other two pooled positions are selected from the group ofthree pools consisting of (1) M or K, (2) R or Y and (3) W or S, wherethe single letters are IUPAC standard ambiguity codes. The sequence of apooled probe is thus, of the form XXXN [(M/K) or (R/Y) or (W/S)].[(M/K)or (R/Y) or (W/S)]XXXXX, where XXX represents bases complementary to thereference sequence. The three pooled positions may be in any order, andmay be contiguous or separated by intervening nucleotides. For, the twopositions occupied by [(M/K) or (R/Y) or (W/S)], two choices must bemade. First, one must select one of the following three pairs of poolednucleotides. (1) M/K, (2) R/Y and (3) W/S. The one of three poolednucleotides selected may be the same or different at the two pooledpositions. Second, supposing, for example, one selects M/K at oneposition, one must then choose between M or K. This choice should resultin selection of a pooled nucleotide comprising a nucleotide thatcomplements the corresponding nucleotide in a reference sequence, whenthe probe and reference sequence are maximally aligned. The sameprinciple governs the selection between R and Y, and between W and S. Atrellis pool probe has one pooled position with four possibilities, andtwo pooled positions, each with two possibilities. Thus, a trellis poolprobe comprises a mixture of 16 (4×2×2) probes. Since each pooledposition includes one nucleotide that complements the correspondingnucleotide from the reference sequence, one of these 16 probes has asegment that is the exact complement of the reference sequence. A targetsequence that is the same as the reference sequence (i.e., a wildtypetarget) gives a hybridization signal to each probe cell. Here, as inother tiling methods, the segment of complementarity should besufficiently long to permit specific hybridization of a pooled probe toa reference sequence be detected relative to a variant of that referencesequence. Typically, the segment of complementarity-is about is 9-21nucleotides.

A target sequence is analyzed by comparing hybridization intensities atthree pooled probes, each having the structure described above. Thesegments complementary to the reference sequence present in the threepooled probes show some overlap. Sometimes the segments are identical(other than at the interrogation positions). However, this need not bethe case. For example, the segments can tile across a reference sequencein increments of one nucleotide (i.e., one pooled probe differs from thenext by the acquisition of one nucleotide at the 5′ end and loss of anucleotide at the 3′ end). The three interrogation positions may or maynot occur at the same relative positions within each pooled probe (i.e.,spacing from a probe terminus). All that is required is that one of thethree interrogation positions from each of the three pooled probesaligns with the same nucleotide in the reference sequence, and that thisinterrogation position is occupied by a different pooled nucleotide ineach of the three probes. In one of the three probes, the interrogationposition is occupied by an N. In the other two pooled probes theinterrogation position is occupied by one of (M/K) or (R/Y) or (W/S).

In the simplest form of the trellis strategy, three pooled probes areused to analyze a single nucleotide in the reference sequence. Muchgreater economy of probes is achieved when more pooled probes areincluded in an array. For example, consider an array of five pooledprobes each having the general structure outlined above. Three of thesepooled probes have an interrogation position that aligns with the samenucleotide in the reference sequence and are used to read thatnucleotide. A different combination of three probes have aninterrogation position that aligns with a different nucleotide in thereference sequence. Comparison of these three probe intensities allowsanalysis of this second nucleotide. Still another combination of threepooled probes from the set of five have an interrogation position thataligns with a third nucleotide in the reference sequence and, theseprobes are used to analyze that nucleotide. Thus, three nucleotides inthe reference sequence are fully analyzed from only five pooled probes.By comparison, the basic tiling strategy would require 12 probes for asimilar analysis.

As an example, a pooled probe for analysis of a target sequence by thetrellis strategy is shown below: Target: ATTAACCACTCACGGGAGCTCT Pool:TGGTGNKYGCCCT

The pooled probe actually comprises 16 individual probes: TGGTGAGcGCCCT+TGGTGcGcGCCCT +TGGTGgGcGCCCT +TGGTGtGcGCCCT +TGGTGAtcGCCCT+TGGTGctcGCCCT +TGGTGgtcGCCCT +TGGTGttcGCCCT +TGGTGAGTGCCCT+TGGTGcGTGCCCT +TGGTGgGTGCCCT +TGGTGtGTGCCCT +TGGTGAtTGCCCT+TGGTGctTGCCCT +TGGTGgtTGCCCT +TGGTGttTGCCCT

The trellis strategy employs an array of probes having at least threecells, each of which is occupied by a pooled probe as described above.Consider the use of three such pooled probes for analyzing a targetsequence, of which one position may contain any single base substitutionto the reference sequence (i.e., there are four possible targetsequences to be distinguished). Three cells are occupied by pooledprobes having a pooled interrogation position corresponding to theposition of possible substitution in the target sequence, one cell withan ‘N’, one cell with one of ‘M’ or ‘K’, and one cell with ‘R’ or ‘Y’.An interrogation position corresponds to a nucleotide in the targetsequence if it aligns adjacent with that nucleotide when the probe andtarget sequence are aligned to maximize complementarity. Note thatalthough each of the pooled probes has two other pooled positions, thesepositions are not relevant for the present illustration. The positionsare only relevant when more than one position in the target sequence isto be read, a circumstance that will be considered later. For presentpurposes, the cell with the ‘N’ in the interrogation position lights upfor the wildtype sequence and any of the three single base substitutionsof the target sequence. The cell with M/K in the interrogation positionlights up for the wildtype sequence and one of the single-basesubstitutions. The cell with R/Y in the interrogation position lights upfor the wildtype sequence and a second of the single-base substitutions.Thus, the four possible target sequences hybridize to the three pools ofprobes in four distinct patterns, and the four possible target sequencescan be distinguished.

To illustrate further, consider four possible target sequences(differing at a single position) and a pooled probe having three pooledpositions, N, K and Y with the Y position as the interrogation position(ie., aligned with the variable position in the target sequence): TargetWild: ATTAACCACTCACGGGAGCTCT (w) Mutants: ATTAACCACTCcCGGGAGCTCT (c)Mutants: ATTAACCACTCgCGGGAGCTCT (g) Mutants: ATTAACCACTCtCGGGAGCTCT (t)TGGTGNKYGCCCT (pooled probe).

The sixteen individual component probes of the pooled probe hybridize tothe four possible target sequences as follows: TARGET w c g tTGGTGAGcGCCCT n n y n TGGTGcGcGCCCT n n n n TGGTGgGcGCCCT n n n nTGGTGtGcGCCCT n n n n TGGTGAtcGCCCT n n n n TGGTGctcGCCCT n n n nTGGTGgtcGCCCT n n n n TGGTGttcGCCCT n n n n TGGTGAGTGCCCT y n n nTGGTGcGTGCCCT n n n n TGGTGgGTGCCCT n n n n TGGTGtGTGCCCT n n n nTGGTGAtTGCCCT n n n n TGGTGctTGCCCT n n n n TGGTGgtTGCCCT n n n nTGGTGttTGCCCT n n n n

The pooled probe hybridizes according to the aggregate of itscomponents: Pool: TGGTGNKYGCCCT y n y nthus, as stated above, it can be seen that a pooled probe having a y atthe interrogation position hybridizes to the wildtype target and one ofthe mutants. Similar tables can be drawn to illustrate the hybridizationpatterns of probe pools having other pooled nucleotides at theinterrogation position.

The above strategy of using pooled probes to analyze a single base in atarget sequence can readily be extended to analyze any number of bases.At this point, the purpose of including three pooled positions withineach probe will become apparent. In the example that follows, ten poolsof probes, each containing three pooled probe positions, can be used toanalyze a each of a contiguous sequence of eight nucleotides in a targetsequence. ATTAACCACTCACGGGAGCTCT Reference sequence -------- Readablenucleotides Pools:  4 TAATTNKYGAGTG  5  AATTGNKRAGTGC  6   ATTGGNKRGTGCC 7    TTGGTNMRTGCCC  8     TGGTGNKYGCCCT  9      GGTGANKRCCCTC 10      GTGAGNKYCCTCG 11        TGAGTNMYCTCGA 12         GAGTGNMYTCGAG 13         AGTGCNMYCGAGA

In this example, the different pooled probes tile across the referencesequence, each pooled probe differing from the next by increments of onenucleotide. For each of the readable nucleotides in the referencesequence, there are three probe pools having a pooled interrogationposition aligned with the readable nucleotide. For example, the 12thnucleotide from the left in the reference sequence is aligned withpooled interrogation positions in pooled probes 8, 9, and 10. Comparisonof the hybridization intensities of these pooled probes reveals theidentity of the nucleotide occupying position 12 in a target sequence.Pools Targets 8 9 10 Wild: ATTAACCACTCACGGGAGCTCT Y Y Y Mutants:ATTAACCACTCcCGGGAGCTCT N Y Y Mutants: ATTAACCACTCgCGGGAGCTCT Y N YMutants: ATTAACCACTCtCGGGAGCTCT N N Y

Example Intensities:

Thus, for example, if pools 8, 9 and 10 all light up, one knows thetarget sequence is wildtype. If pools, 9 and 10 light up, the targetsequence has a C mutant at position 12. If pools 8 and 10 light up, thetarget sequence has a G mutant at position 12. If only pool 10 lightsup, the target sequence has a t mutant at position 12. and 10 light up,the target sequence has a C mutant at position 12. If pools 8 and 10light up, the target sequence has a G mutant at position 12. If onlypool 10 lights up, the target sequence has a t mutant at position 12.

The identity of other nucleotides in the target sequence is determinedby a comparison of other sets of three pooled probes. For example, theidentity of the 13th nucleotide in the target sequence is determined bycomparing the hybridization patterns of the probe pools designated 9, 10and 11. Similarly, the identity of the 14th nucleotide in the targetsequence is determined by comparing the hybridization patterns of theprobe pools designated 10, 11, and 12.

In the above example, successive probes tile across the referencesequence in increments of one nucleotide, and each probe has threeinterrogation positions occupying the same positions in each proberelative to the terminus of the probe (i.e., the 7, 8 and 9th positionsrelative to the 3′ terminus). However, the trellis strategy does notrequire that probes tile in increments of one or that the interrogationposition positions occur in the same position in each probe. In avariant of trellis tiling referred to as “loop” tiling, a nucleotide ofinterest in a target sequence is read by comparison of pooled probes,which each have a pooled interrogation position corresponding to thenucleotide of interest, but in which the spacing of the interrogationposition in the probe differs from probe to probe. Analogously to theblock-tiling approach, this allows several nucleotides to be read from atarget sequence from a collection of probes that are identical except atthe interrogation position. The identity in sequence of probes,particularly at their 3′ termini, simplifies synthesis of the array andresult in more uniform probe density per cell.

To illustrate the loop strategy, consider a reference sequence of whichthe 4, 5, 6, 7 and 8th nucleotides (from the 3′ termini are to be read.All of the four possible nucleotides at each of these positions can beread from comparison of hybridization intensities of five pooled probes.Note that the pooled positions in the probes are different (for examplein probe 55, the pooled positions are 4, 5 and 6 and in probe 56, 5, 6and 7). TAACCACTCACGGGAGCA Reference sequence 55 ATTNKYGAGTGCC 56ATTGNKRAGTGCC 57 ATTGGNKRGTGCC 58 ATTRGTNMGTGCC 59 ATTKRTGNGTGCC

Each position of interest in the reference sequence is read by comparinghybridization intensities for the three probe pools-that have aninterrogation position aligned with the nucleotide of interest in thereference sequence. For ;example, to read the fourth nucleotide in thereference sequence, probes 55, 58 and 59 provide pools at the fourthposition. Similarly, to read the fifth nucleotide in the referencesequence, probes 55, 56 and 59 provide pools at the fifth position. Asin the previous trellis strategy, one of the three probes being comparedhas an N at the pooled position and-the other two have M or K, and (2) Ror Y and (3) W or S.

The hybridization pattern of the five pooled probes to target sequencesrepresenting each possible nucleotide substitution at five positions inthe reference sequence is shown below. Each possible substitutionresults in a unique hybridization pattern at three pooled probes, andthe identity of the nucleotide at that position can be deduced from thehybridization pattern. Pools Targets 55 56 57 58 59 Wild:TAACCACTCACGGGAGCA Y Y Y Y Y Mutant: TAAgCACTCACGGGAGCA Y N N N NMutant: TAAtCACTCACGGGAGCA Y N N Y N Mutant: TAAaCACTCACGGGAGCA Y N N NY Mutant: TAACgACTCACGGGAGCA N Y N N N Mutant: TAACtACTCACGGGAGCA N Y NN Y Mutant: TAACaACTCACGGGAGCA Y Y N N N Mutant: TAACCcCTCACGGGAGCA N YY N N Mutant: TAACCgCTCACGGGAGCA Y N Y N N Mutant: TAACCtCTCACGGGAGCA NN Y N N Mutant: TAACCAgTCACGGGAGCA N N N Y N Mutant: TAACCAtTCACGGGAGCAN Y N Y N Mutant: TAACCAaTCACGGGAGCA N N Y Y N Mutant:TAACCACaCACGGGAGCA N N N N Y Mutant: TAACCACcCACGGGAGCA N N Y N YMutant: TAACCACgCACGGGAGCA N N N Y Y

Many variations on the loop and trellis tilings can be created. All thatis required is that each position in sequence must have a probe with a‘N’, a probe containing one of R/Y, M/K or W/S, and a probe containing adifferent pool from that set, complementary to the wild type target atthat position, and at least one probe with no pool at all at thatposition. This combination allows all mutations at that position to beuniquely detected and identified.

A further class of strategies involving pooled probes are termed codingstrategies. These strategies assign code words from some set of numbersto variants of a reference sequence. Any number of variants can becoded. The variants can include multiple closely spaced substitutions,deletions or insertions. The designation letters or other symbolsassigned to each variant may be any arbitrary set of numbers, in anyorder. For example, a binary code is often used, but codes to otherbases are entirely feasible. The numbers are often assigned such thateach variant has a designation having at least one digit and at leastone nonzero value for that digit. For example, in a binary system, avariant assigned the number 101, has a designation of three digits, withone possible nonzero value for each digit.

The designation of the variants are coded into an array of pooled probescomprising a pooled probe for each nonzero value of each digit in thenumbers assigned to the variants. For example, if the variants areassigned successive number in a numbering system of base m, and thehighest number assigned to a variant has n digits, the array would haveabout n×(m−1) pooled probes. In general, log_(m) (3N+1) probes arerequired to analyze all variants of N locations in a reference sequence,each having three possible mutant substitutions. For example, 10 basepairs of sequence may be analyzed with only 5 pooled probes using abinary coding system.

Each pooled probe has a segment exactly complementary to the referencesequence except that certain positions are pooled. The segment should besufficiently long to allow specific hybridization of the pooled probe tothe reference sequence relative to a mutated form of the referencesequence. As in other tiling strategies, segments lengths of 9-21nucleotides are typical. Often the probe has no nucleotide's other thanthe 9-21 nucleotide segment. The pooled positions comprise nucleotidesthat allow the pooled probe to hybridize to every variant assigned aparticular nonzero value in a particular digit.. Usually, the pooledpositions further comprises a nucleotide that allows the pooled probe tohybridize to the reference sequence. Thus, a wildtype target (orreference sequence) is immediately recognizable from all the pooledprobes being lit.

When a target is hybridized to the pools, only those pools comprising acomponent probe having a segment that is exactly complementary to thetarget light up. The identity of the target is then decoded from thepattern of hybridizing pools. Each pool that lights up is correlatedwith a particular value in a particular digit. Thus, the aggregatehybridization patterns of each lighting pool reveal the value of eachdigit in the code defining the identity of the target hybridized to thearray.

As an example, consider a reference sequence having four positions, eachof which can be occupied by three possible mutations. Thus, in totalthere are 4×3 possible variant forms of the reference sequence. Eachvariant is assigned a binary number 0001-1100 and the wildtype referencesequence is assigned the binary number 1111. Positions X X X X - 4Target: TAACCACGGGAGCA C = 1111 A = 1111 C = 1111 T = 1111 G = 0001 C =0010 G = 0011 A = 0100 T = 0101 G = 0110 T − 0111 C = 1000 A = 1001 T =1010 A = 1011 G = 1100

A first pooled probe is designed by including probes that complementexactly each variant having a 1 in the first digit. target(1111): TAAC CA C T CACGGGAGCA Mutant(0001): TAAC  g A C T CACGGGAGCA Mutant(0101):TAAC   t A C T CACGGGAGCA Mutant(1001): TAAC    a A C T CACGGGAGCAMutant(0011): TAAC  C A  g T CACGGGAGCA Mutant(0111): TAAC  C A   t TCACGGGAGCA Mutant(1101): TAAC  C A    a T CACGGGAGCA First pooled probe= ATTG [GCAT] T [GCAT] A GTGCCC = ATTG N T N A GTGCCC

Second, third and fourth pooled probes are then designed respectivelyincluding component probes that hybridize.to each variant having a 1 inthe second, third and fourth digit. XXXX - 4 positions examined Target:TAACCACTCACGGGAGCA Pool 1(1): ATTGnTnAGTGCCC = 16 probes (4×1×4×1) Pool2(2): ATTGGnnAGTGCCC = 16 probes (1×4×4×1) Pool 3(4): ATTGyrydGTGCCC =24 probes (2×2×2×3) Pool 4(8): ATTGmwmbGTGCCC = 24 probes (2×2×2×3)

The pooled probes hybridize to variant targets as follows: Pools Targets1 2 3 4 Wild(1111) TAACCACTCACGGGAGCA Y Y Y Y Mutant(0001):TAACgACTCACGGGAGCA Y N N N Mutant(0101): TAACtACTCACGGGAGCA Y N Y NMutant(1001): TAACaACTCACGGGAGCA Y N N Y Mutant(0010):TAACCcCTCACGGGAGCA N Y N N Mutant(0110): TAACCgCTCACGGGAGCA N Y Y NMutant(1010): TAACCtCTCACGGGAGCA N Y N Y Mutant(0011):TAACCAgTCACGGGAGCA Y Y N N Mutant(0111): TAACCAtTCACGGGAGCA Y Y Y NMutant(1101): TAACCAaTCACGGGAGCA Y N Y Y Mutant(0100):TAACCACaCACGGGAGCA N N Y N Mutant(1000): TAACCACcCACGGGAGCA N N N YMutant(1100): TAACCACgCACGGGAGCA N N Y Y

The identity of a variant (i.e., mutant) target is read directly fromthe hybridization pattern of the pooled probes. For example the mutantassigned the number 0001 gives a hybridization pattern of NNNY withrespect to probes 4, 3, 2 and 1 respectively.

In the above example, variants are assigned successive numbers in anumbering system. In other z,999 odiments, sets of numbers can be chosenfor their properties. If the codewords are chosen from an error-controlcode, the properties of that code carry over to sequence analysis. Anerror code is a numbering system in which some designations are assignedto variants and other designations serve to indicate errors that mayhave occurred in the hybridization process. For example, if allcodewords, have an odd number of nonzero digits (binary coding+errordetection), any single error in hybridization will be detected by havingan even number of pools lit. Wild Target: TAACCACTCACGGGAGCA Pool 1(1):ATTGnAnAGTGCCC = 16 Probes (4×1×4×1) Pool 2(2): ATTGGnnAGTGCCC = 16Probes (1×4×4×1) Pool 3(4): ATTGryrhGTGCCC = 24 Probes (2×2×2×3) Pool4(8): ATTGkwkvGTGCCC = 24 Probes (2×2×2×3)

A fifth probe can be added to make the number of pools that hybridize toany single mutation odd. Pool 5(c): ATTGdhsmGTGCCC = 36 probes (2×2×3×3)Hybridization of pooled probes to targets: Pool Target 1 2 3 4 5Target(11111): TAACCACTCACGGGAGCA Y Y Y Y Y Mutant(00001):TAACgACTCACGGGAGCA Y N N N N Mutant(10101): TAACtACTCACGGGAGCA Y N N N NMutant(11001): TAACaACTCACGGGAGCA Y N N Y Y Mutant(00010):TAACCcCTCACGGGAGCA N Y N N N Mutant(10110): TAACCgCTCACGGGAGCA N Y Y N YMutant(11010): TAACCtCTCACGGGAGCA N Y N Y Y Mutant(10011):TAACCAgTCACGGGAGCA Y Y N N Y Mutant(00111): TAACAtTCACGGGAGCA Y Y Y N NMutant(01101): TAACCAaTCACGGGAGCA Y N Y Y N Mutant(00100):TAACCACaCACGGGAGCA N N Y N N Mutant(01000): TAACCAcCCACGGGAGCA N N N Y NMutant(11100): TAACCACgCACGGGAGCA N N Y Y Y

9. Bridging Strategy

Probes that contain partial matches to two separate (i.e., noncontiguous) subsequences of a target sequence sometimes hybridizestrongly to the target sequence. In certain instances, such probes havegenerated stronger signals than probes of the same length which areperfect matches to the target sequence. It is believed (but notnecessary to the invention) that this observation results frominteractions of a single target sequence with two or more probessimultaneously. This invention exploits this observation to providearrays of probes having at least first and second segments, which arerespectively complementary to first and second subsequences of areference sequence. Optionally, the probes may have a third or morecomplementary segments. These probes can be employed in any of thestrategies noted above. The two segments of such a probe can becomplementary to disjoint subsequences of the reference sequences orcontiguous subsequences. If the latter, the two segments in the probeare inverted relative to the order of the complement of the referencesequence. The two subsequences of the reference sequence each typicallycomprises about 3 to 30 contiguous nucleotides. The subsequences of thereference sequence are sometimes separated by 0, 1, 2 or 3 bases. Oftenthe sequences, are adjacent and nonoverlapping.

For example, a wildtype probe is created by complementing two sectionsof a reference sequence (indicated by subscript and superscript) andreversing their order. The interrogation position is designated (*) andis apparent from comparison of the structure of the wildtype probe withthe three mismatched probes. The corresponding nucleotide in thereference sequence is the “a” in the superscripted segment. Reference:5′ T_(GGCTA) ^(CGAGG)AATCATCTGTTA      * Probes: 3′ GCTCC CCGAT (Probefrom first probe set) 3′ GCACC CCGAT 3′ GCCCC CCGAT 3′ GCGCC CCGAT Theexpected hybridizations are: Match: GCTCCCCGAT ...TGGCTACGAGGAATCATCTGTTA        GCTCCCCGAT Mismatch: GCTCCCCGAT ...TGGCTACGAGGAATCATCTGTTA         GCGCCCCGAT

Bridge tilings are specified using a notation which gives the length ofthe two constituent segments and the relative position of theinterrogation position. The designation n/m indicates a segmentcomplementary to a region of the reference sequence which extends for nbases and is located such that the interrogation position is in the mthbase from the 5′ end. If m is larger than n, this indicates that theentire segment is to the 5′ side of the interrogation position. If m isnegative, it indicates that the interrogation position is the absolutevalue of m bases 5′ of the first base of the segment (m cannot be zero).Probes comprising multiple segments, such as n/m+a/b+ . . . have a firstsegment at the 3′ end of the probe and additional segments added 5′ withrespect to the first segment. For example, a 4/8 tiling consists of(from the 3′ end of the probe) a 4 base complementary segment, starting7 bases 5′ of the interrogation position, followed by a 6 base region inwhich the interrogation position is located at the third base. Betweenthese two segments, one base from the reference sequence is omitted. Bythis notation, the set shown above is a 5/3+⅝ tiling. Many differenttilings are possible with this method, since the lengths of bothsegments can be varied, as well as their relative position (they may bein either order and there may be a gap between them) and their locationrelative to the interrogation position.

As an example, a 16 mer oligo target was hybridized to a chip containingall 4¹⁰ probes of length 10. The chip includes short tilings of bothstandard and bridging types. The data from a standard 10/5 tiling wascompared to data from a 5/3⅝ bridge tiling (see Table 1). Probeintensities (mean count/pixel) are displayed along with discriminationratios (correct probe intensity/highest incorrect probe intensity).Missing intensity values are less than 50 counts. Note that for eachbase displayed the bridge tiling has a higher discrimination value.TABLE 1 Comparison of Standard and Bridge Tilings CORRECT PROBE BASETILING PROBE BASE: C A C C STANDARD A 92 496 294 299 (10/5) C 536 148532 534 G 69 167 72 52 T 146 95 212 126 DISCRIMINATION: 3.7 3.0 1.8 1.8BRIDGING A — 404 — 156 5/3 + 5/8 C 276 — 345 379 G — 80 — — T — — — 58DISCRIMINATION: >5.5 5.1 2.4 1.26

The bridging strategy offers the following advantages:

(1) Higher discrimination between matched and mismatched probes,

(2) The possibility of using longer probes in a bridging tiling, therebyincreasing the specificity of the hybridization, without sacrificingdiscrimination,

(3) The use of probes in which an interrogation position is located veryoff-center relative to the regions of target complementarity. This maybe of particular advantage when, for example, when a probe centeredabout one region of the target gives low hybridization signal. The lowsignal is overcome by using a probe centered about an adjoining regiongiving a higher hybridization signal.

(4) Disruption of secondary structure that might result in annealing ofcertain probes (see previous discussion of helper mutations).

10. Deletion Tiling

Deletion tiling is related to both the bridging and helpermutant.strategies described above. In the deletion strategy, comparisonsare performed between probes sharing a common deletion but differingfrom each other at an interrogation position located outside thedeletion. For example, a first probe comprises first and secondsegments, each exactly complementary to respective first and secondsubsequences of a reference sequence, wherein the first and secondsubsequences of the reference sequence are separated by a short distance(e.g., 1 or 2 nucleotides). The order of the first and second segmentsin the probe is usually the same as that of the complement to the firstand second subsequences in the reference sequence. The interrogationposition is usually separated from The comparison is performed withthree other probes, which are identical to the first probe except at aninterrogation position, which is different in each probe. Reference: . .. AGTACCAGATCTCTAA . . . Probe set: CATGGNC AGAGA (N=interrogationposition).

Such tilings sometimes offer superior discrimination in hybridizationintensities between the probe having an interrogation positioncomplementary to the target and other probes. Thermodynamically, thedifference between the hybridizations to matched and mismatched targetsfor the probe set shown above is the difference between a single-basebulge, and a large asymmetric loop (e.g., two bases of target, one ofprobe). This often results in a larger difference in stability than thecomparison of a perfectly matched probe with a probe showing a singlebase mismatch in the basic tiling strategy.

The superior discrimination offered by deletion tiling is illustrated byTable 2, which compares hybridization data from a standard 10/5 tilingwith a ( 4/8+ 6/3) deletion tiling of the reference sequence. (Thenumerators indicate the length of the segments and the denominators, thespacing of the deletion from the far termini of the segments.) Probeintensities (mean count/pixel) are displayed along with discriminationratios (correct probe intensity/highest incorrect probe intensity). Notethat for each base displayed the deletion tiling has a higherdiscrimination value than either standard tiling shown. TABLE 2Comparison of Standard and Deletion Tilings CORRECT PROBE BASE TILINGPROBE BASE: C A C C STANDARD A 92 496 294 299 (10/5) C 536 148 532 534 G69 167 72 52 T 146 95 212 126 DISCRIMINATION: 3.7 3.0 1.8 1.8 DELETION A6 412 29 48 4/8 + 6/3 C 297 32 465 160 G 8 77 10 4 T 8 26 31 5DISCRIMINATION: 37.1 5.4 15 3.3 STANDARD A 347 533 228 277 (10/7) C 729194 536 496 G 232 231 102 89 T 344 133 163 150 DISCRIMINATION: 2.1 2.32.3 1.8

The use of deletion or bridging probes is quite general. These probescan be used in any of the tiling strategies of the invention. As well asoffering superior discrimination, the use of deletion or bridgingstrategies is advantageous for certain probes to avoidself-hybridization (either within a probe or between two probes of thesame sequence)

11. Nucleotide Repeats

Recently a new form of human mutation, expansion of trinucleotiderepeats, has been found to cause the diseases of fragile X-syndrome,spinal and bulbar atrophy, myotonic dystrophy and Huntington's disease..See Ross et al., TINS 16, 254-259 (1993). Long lengths of trinucleotiderepeats are associated with the mutant form of a gene. The longer thelength, the more severe the consequences of the mutation and the earlierthe age of onset. The invention provides arrays and methods foranalyzing the length of such repeats.

The different probes in such an array comprise different numbers ofrepeats of the complement of the trinucleotide repeat of interest. Forexample, one probe might be a trimer, having one copy of the repeat, asecond probe might be a sixmer, having two copies of the repeat, and athird probe might be a ninmer having three copies, and so forth. Thelargest probes can have up to about sixty bases or 20 trinucleotiderepeats.

The hybridization signal of such probes to a target of trinucleotiderepeats is related to the length of the target. It has been found thaton increasing the target size up to about the length of the probe, thehybridization signal shows a relatively large increase for each completetrinucleotide repeat unit in the target, and a small increase for eachadditional base in the target that does not complete a trinucleotiderepeat. Thus, for example, the hybridization signals for differenttarget sizes to a 20 mer probe show small increases as the target sizeis increased from 6-8 nucleotides and a larger increase as the targetsize is increased to 9 nucleotides.

Arrays of probes having different numbers of repeats are usuallycalibrated using known amounts of target of different length. For eachtarget of known length, the hybridization intensity is recorded for eachprobe. Thus, each target size is defined by the relative hybridizationsignals of a series of probes of different lengths. The array is thenhybridized to an unknown target sequence and the relative hybridizationsignals of the different sized probes are determined. Comparison of therelative hybridization intensity profile for different probes withcomparable data for targets of known size allows interpolation of thesize of the unknown target. Optionally, hybridization of the unknowntarget is performed simultaneously with hybridization of a target ofknown size labelled with a different color.

C. Preparation of Taraet Samples

The target polynucleotide, whose sequence is to be determined, isusually isolated from a tissue sample. If the target is genomic, thesample may be from any tissue (except exclusively red blood cells). Forexample, whole blood, peripheral blood lymphocytes or PBMC, skin, hairor semen are convenient sources of clinical samples. These sources arealso suitable if the target is RNA. Blood and other body fluids are alsoa convenient source for isolating viral nucleic acids. If the target ismRNA, the sample is obtained from a tissue in which the mRNA isexpressed. If the polynucleotide in the sample is RNA, it is usuallyreverse transcribed to DNA. DNA samples or cDNA resulting from reversetranscription are usually amplified, e.g., by PCR. Depending on theselection of primers and amplifying enzyme(s), the amplification productcan be RNA or DNA. Paired primers are selected to flank the borders of atarget polynucleotide of interest. More than one target can besimultaneously amplified by multiplex PCR in which multiple pairedprimers are employed. The target can be labelled at one or morenucleotides during or after amplification. For some targetpolynucleotides (depending on size of sample), e.g., episomal DNA,sufficient DNA is present in the tissue sample to dispense with theamplification step.

When the target strand is prepared in single-stranded form as inpreparation of target RNA, the sense of the strand should of course becomplementary to that of the probes on the chip. This is achieved byappropriate selection of primers. The target is preferably fragmentedbefore application to the chip to reduce or eliminate the formation ofsecondary structures in the target. The average size of targets segmentsfollowing hybridization is usually larger than the size of probe on thechip.

II. Biotransformation Gene Chips

A. Biotransformation Genes

Biotransformation genes tiled by the invention include any of the 481known cytochrome P450 genes, particularly, the human P450 genes (seeNebert, DNA & Cell Biol. 10, 1-14 (1991); Nelson et al.,Pharmacogenetics 6, 1-42 (1996), acetylase genes, monoamine oxidasegenes, and genes known to specifically biotransform particular drugs,such as the gene encoding glucuronidase that participates in the pathwayby which codeine or morphine are converted to active form. Paul et al.,J. Pharm. Exp. Ther. 251, 477 (1989). Other genes of particular interestinclude P450 2D6, P450 2C19, N-acetyl transferase II, glucose6-phosphate dehydrogenase, pseudocholinesterase, catechol-O-methyltransferase, thiopurine methyltransferase and dihydropyridinedehydrogenase. cDNA and at least partial-genomic DNA sequences areavailable for these genes, e.g., from data bases such as GenBank andEMBL (see Table 3). TABLE 3 ACCESSION NUMBER CYP LIST ACCESSION GENENUMBER(S) IMPORTANCE CYP1A1 D12525 Cancer Susceptibility D01198 CYP1A2M31664 M31665 M31666 M31667 U02993 CYP2A X13897 CYP2A3 M33318 Coumarin7-hydroxylation M33316 CYP2A4 X13930 CYP2C8 X54807 X54808 CYP2C9 M61855Warfarin Metabolism J05326 M61857 J05326 L16877 CYP2C17 M61858 J05326CYP2C18 M61853 Drug Metabolism J05326 M61856 CYP2C19 L07093S-mephenytoin 4-hydroxylase M61854 J05326 M15331 CYP2D6 M20403Debrisoquin/Sparteine Polymorphism M19697 M24499 X16866 X58467 CYP2D7Ppseudogene X58468 CYP2D8P pseudogene CYP2E1 D10014 Ethanol InducibleJ02843 CYP3A4 D11131 Polymorphic Drug Metabolism M14096 CYP4F2 U02388Leukotriene B4 omega hydroxylase NAT2 U23052 Drug Acetylation/DrugInduced Disease U23434 TPMT U11424 Thiopurine Methyl Transferase-transplantation and childhood leukemia U12387

Additional genomic sequence flanking the regions already sequenced areeasily determined by PCR-based gene walking. See Parker et al., Nucl.Acids Res. 19:3055-3060. A specific primer for the sequenced region isprimed with a general primer that hybridizes to the flanking region.

The CYP2D6 enzyme has debrisoquine oxidase activity. See e.g., Kimura etal., Am. J. Human. Genet. 45, 889-904 (1989).

Several therapeutically important compounds are metabolized by CYP2D6.The list includes cardioactive drugs: β-blockers (bufuralol,propranolol, metoprolol, timolol) and. antiarrhythmics (sparteine,encainide, flecainide, mexiletine) (Buchert & Woosley, Pharmacogenetics2, 2-11 (1992); Birgersdotter et al., Brit. J. Clin. Pharmacol. 33,275-280 (1992)); psychoactive drugs including tricyclic antidepressants(imipramine, desipramine, nortriptyline) and antipsychotics (clozapineand haloperidol) (Dahl & Bertilsson, Pharmacogenetics 3, 61-70 (1993);Fischer et al., J. Pharmacol. Exp. Ther. 260, 1355-1360 (1992); Lerenaet al., Drug Monitor 14, 92-97 (1992)); as well as a variety of othercommonly used drugs including codeine and dextromethorphan (Eichelbaum &Gross, Pharmac. Ther. 46, 377-394 (1990)) as well as amphetamine, andcocaine. Ten percent of the general population is defective in P450 2D6,an enzyme that demethethylates codeine at an earlier stage in theactivation pathway, and therefore derives no analgesic benefit fromcodeine (see Sindrup & Brosen, Pharmacogenetics 5, 335-346 (1995))

At least seven different polymorphic variants of the CYP2D6 genedemonstrating autosomal recessive inheritance are associated with a poordrug metabolizer phenotype (see Table 4). These alleles are designatedCYP2D6A, CYP2D6B, CYP2D6C, CYP2D6D, CYP2D6E, CYP2D6F, and CYP2D6J(Gonzales & Idle, Clin. Pharmacokinet. 26(1), 59-70 (1994); Nelson etal., DNA & Cell Biol. 12(1), 1-51 (1993)). CYP2D6A, CYP2D6E and CYP2D6Fare minor variants of the wild type gene. CYP2D6A has a singlenucleotide deletion in exon 5 with a consequent frame shift (Kagimoto etal., J. Biol. Chem. 265, 17209-17214 (1990)). CYP2D6E and CYP2D6F arerare, recently described variants (Gonzales & Idle, supra). CYP2D6Baccounts for about 70% of defective alleles. This variant has pointmutations in exons 1, 3, 8 and 9 as well as a base change at the thirdintron splice site that results in aberrant transcript splicing(Gonzales et al., Nature 331, 442-446 (1988); Kagimoto et al., J. Biol.Chem. 265, 17209-17214 (1990)). CYP2D6C has a three base deletion inexon 5 (Broly and Meyer, Pharmacogenetics 3, 123-130 (1993)) and, on theCYP2D6D allele, the entire functional gene is deleted although thepseudogenes remain intact (Gaedigk et al., Am. J. Hum. Genet. 48,943-950 (1991)). The CYP2D6J allele has base changes in both the firstand ninth exons that result in amino acid changes (Yokota et al.,Pharmacogenetics 3, 256-263 (1993). The CYP2D6 gene clusters with otherCYP2D genes on human chromosome 22. Also present in this region are twoor three highly conserved pseudogenes. Of these, CYP2D7P (three variantforms) and CYP2D8P have been isolated and sequenced (Kimura et al.,supra; Helm & Meyer, supra). TABLE 4 (EXON) XBAI NUCLEOTIDE HAP- ENZYMEALLELE CHANGES LOTYPE ACTIVITY REF. CYP2D6-wt 29 kb NORMAL (97) (9)CYP2D6-LI (3) 1726 G → C 29 kb (12) (6) 2938 C → (11) T/296 Arg → Cys(13) (9) 4268 G → C/486 Ser → Thr (6) 2938 C → T/296 Arg → Cys (6) 2938C → T/296 Arg → Cys CYP2D6-A (5) 2637 ΔA 29 kb ABSENT (15) CYP2D6-B (4)1934A (+6 29 kb (15) other mutations) 44 kb (14) 9 + 16 kb (99) CYP2D6-DDeletion 11.5 kb (13 kb) CYP2D6-E (6) 3023 A → 29 kb C/324 His → ProCYP2D6- (3) 1795 ΔT/ 29 kb (98, ΔT1795 152 Try → Gly 100) 153 StopCYP2D6-C (5) 2703-5 ΔAAG/ 29 kb DE- (44) 281 ΔLys CREASED (101) CYP2D6-J(1) 188 C → 29 kb (16) T/Pro 34 Pro → Ser 44 kb (3) 1749 G → C (9) 4268G → C/486 Ser → Thr CYP2D6-W (1) 188 C → T/34 29 kb (102) Pro → Ser 44kb (9) 4268 G → C/486 Ser → Thr CYP2D6- (1) 188 C → T/34 29 kb (103) Ch1Pro → Ser 44 kb (2) 1127 C → T (3) 1749 G → C (9) 4268 G → C/486 Ser →Thr (CYP2D6- Amplification of 175 kb IN- (12) L)₁₂ D6-L 42 kb CREASED(12) (CYP2D6- Duplication of D6-L L)₂Presently used trivial names of CYP2D6 alleles, summary ofCYP2D6-Alleles, haplotypes and their phenotypic consequences (modifiedfrom U. A. Meyer).(9) Kimura et al. Am J Hum Genet 45: 889-904 (1989)(11) Armstrong et al. Hum Genet 91: 616-617 (1993)(12) Johansson et al. PNAS 90: 11825-11829 (1993)(13) Tsuneoka et al. J. Biochem Tokyo 114: 263-266 (1993)(14) Gaedigk et al. Am J Hum Genet 48: 943-950 (1991)(15) Kagimoto et al. J Biol Chem 265: 17209-17214 (1990)(16) Yokota et al. Pharmacogenet 3: 256-263 (1993)(44) Tyndale et al. Pharmacogenet 1: 26-32 (1991)(97) Gonzales et al. Nature 331: 442-446 (1988)(98) Evert et al. Pharmacogenet 4: 271-274 (1994)(99) Evert et al. Naunyn-Schmiedebergs Arch Pharmacol 350: 434-439(1994)(100) Saxena et al. Hum Mol Genet 3: 923-926 (1994)(101) Broly et al. Pharmacogenet 3: 123-130 (1993)(102) Wang et al. Clin Pharmacol Ther 53: 410-418 (1993)(103) Johansson et al. Mol Pharmacol 46: 452-459 (1994)The 2C19 gene is the principal human determinant of S-mephenytoinhydroxylase. Drugs metabolized by this enzyme in addition to mephenytoininclude antidepressants and neuroleptics. Variant alleles are describedin de Morais et al., J. Biol. Chem. 269(22), 15419-15422 (1994);# de Morais et al., Molecular Pharmacology 46, 594-598 (1994). Mutationsare known to occur at nucleotides 636 (G-A) and 681 (G-A) of the codingsequence.CYP2E1 is responsible for metabolizing several anesthetics includingethanol.CYP2A6 metabolizes nicotine.CYP2C9 metabolizes warfarin. A table showing other pairs of drugs andcytochromes P450 that either metabolize the drug or are inhibited by itappears below.

TABLE 5 Cytochrome P450 (CYP) Isoenzyme Inhibited Drug ClassMetabolizing Enzymes Enzymes Comments Roxithromycin Antibiotic 3A4 CinPharm and Ther 1991, 49, 158 Spiramycin Antibiotic 3A4 Cin Pharm andTher 1991, 49, 158 Taxol Antitumor 6a-hydroxylation - 3A J Pharm ExpTher 1994, 268, 1160-1165 Tiracizine Antiarrhythmic Urethane cleavage -Abstracts - 10th Int Symp 2D6 Mic & Drug Oxid 1994, p 590 TrimipramineAntidepressant Hydroxylation - 2D6 Chem Path Pharm 1993, 82, 111-120Tropisetron 5-HT3 5,6,7-hydroxylation - Drug Met Disp 1994, 22,antogonist 2D6 269-274 Zanoterone Anticancer Hydroxylation - 3A4/5Abstracts - 10th Int Symp Mic & Drug Oxid 1994, p 593 EconazoleAntifungal 3A4 > 1A2 > 2C, Clin Pharm and Ther 1991, 2D6 49, 158(abstract P11-37) Ethosuximide Anticonvulsant 3A Xenobiotic 1993, 23,307-315 Finasteride 5α-Reductase 3A4 Abstracts - 10th Int Symp InhibitorMic & Drug Oxide 1994, p 594 FK 506 Immunosuppressant 3A4 (major), 2D6Abstracts - 10th Int Symp (<10%) Mic & Drug Oxid 1994, p 587 FlexerilMuscle N-demethylation - Abstracts - 10th Int Symp relaxant 1A2, 3A4,2D6 (minor) Mic & Drug Oxid 1994, p 592 Haloperidol Neuroleptic 3AAbstracts - 10th Int Symp Agent Mic & Drug Oxid 1994, p 179 IbuprofenNSAID 2C8, 2C9, 2C18 Clin Pharmacokinetics 1994, 26, 59-70 IfosfamideAnticancer 4-hydroxylation, N- Biochem Pharmacol 1994, 47,dechloroethylation - 1157-1163 3A4 Itraconazole Antifungal 3A4 > 1A2 >2C, Clin Pharm and Ther 1991, 2D6 49, 158 (abstract PII-37) LabetalolAntihypertensive 2D6 Drug Met Disp 1985, 13, 443-448 Ondansetron 5-HT37,8-hydroxylation - Drug Met Disp 1994, 22, antongonist 3A, 2D6 269-274Oxodipine Antihypertensive 3A4 J Pharm Exp Ther 1992, 261, 381-386Prednisolone Corticosteroid 3A4 Drug Met Disp 1990, 18, 595-606Alfentanil Analgesic 3A4 Anesthesiology 1992, 77, 467-474 AmiflamineMAO-A 2D6 Clin Pharmacol Ther 1984, Inhibitor 36, 515-519 AzithromycinAntibiotic 3A4 Clin Pharm and Ther 1991, 49, 158 Benzphetamine Anorectic2C8, 2C9, 2C18, 3A4 Clin Pharmacokinetics 1994, 26, 59-70 CaptoprilAntihypertensive 2D6 Eur J Clin Pharm 1987, 31, 633-641 CitalopramAntidepressant 2C18, 2C19 Ther Drug Monit 1993, 15, 11-17 ClarithromycinAntibiotic 3A4 Clin Pharm and Ther 1991, 49, 158 ClonazepamAnticonvulsant Nitroreduction - 3A4 Fundam Clin Pharm 1993, 7, 69-75Clotramizole Antifungal 3A4 > 1A2 > 2C, Clin Pharm and Ther 1991, 2D649, 158 (abstract PII-37) Cocaine N-demethylation - 3A4 Pharmacol 1993,46, 294-300 Dapsone Antibacterial N-hydroxylation - 3A4 Mol Pharmacol1992, 41, 975-980 Delavirdine HIV-1 Reverse Hydroxylation - 3A4Abstracts - 10th Int Symp transciptase N-dealkylation - 3A4, Mic & DrugOxid 1994, p 240 Inhibitor 2D6 Dextromethorphan AntitussiveO-demethylation - 2D6 Biochem Pharm 1994, 48, N-demethylation - 173-1823A4, 3A5 Diazepam CNS Depressant 2C8, 2C9, 2C18 Clin Pharmacokinetics1994, 26, 59-70 Diclofenac NSAID 2C8, 2C9, 2C18 Clin Pharmacokinetics1994, 26, 59-70 Tamoxifen Antiestrogen 3A4, 1A1 ISSX Proceedings Vol. 3,44 Taxotere Antimitotic 3A ISSX Proceedings Vol. 3, 36 Tenoxicam NSAID2C Life Sci 1992, 51, 575-581 Terfenadine Antihistamine 3A4 Drug MetDisp 1993, 21, 403-409 Timolol β-blocker 2D6 TiPS 1992, 13, 434-439Thioridazine Neuroleptic 2D6 TiPS 1992, 13, 434-439 Tolbutamide Bloodglucose 2C18 lowering agent Tomoxetine Antidepressant 2D6 TiPS 1992, 13,434-439 Toremifene Antiestrogen 3A4/3A5 - (N- ISSX Proceedings Vol 3, 22demethylation), 1A Triazolam Hypnotic 3A4 Trifluperidol Neuroleptic 2D6TiPS 1992, 13, 434-439 Troleandomycin Antibiotic 3A4 VerapamilAntihypertensive 3A4 (mainly), also Arch Pharmacol 1993, 348, 1A2 forD-617 332-337 metabolite Vinblastine Antitumor 3A4 WarfarinAnticoagulant 3A, 2C, 1A2 Zonisamide Anticonvulsant 3A Molec Pharm 1993,44, 216-221 Acetaminophen Antipyretic 3A4, 2E1, 1A2 Chem Res Tox 1993,6, 511 Amiodarone Antiarrhythmic 3A - deethylatin, 1A2 1A2, Drug MetDisp 1993, 21, 2C18, 978-985 2D6, 3A4 Amitriptyline Antidepressant 2C18,2D6 Astemizole Anthistamine 3A4 Bufuralol β-Blocker 2D6 TiPS 1992, 13,434-439 Carbamazepine Anticonvulsant 3A Inducer of 3A4; Clin Pharmacokin1993, 25, 450-482 Chlorzoxazone Muscle 2E1 - 6-OH metabolite Chem ResTox 1990, 3, 566-573 relaxant Cimetidine Antiulcer 3A4, 2D6 > 1A2,Gastroent 1991, 101, 1680-1691 2E1 Ciprofloxacin Antimicrobial 3A4 ClinPharmacokinet 1992, 23, 132-146 Clomipramine Antidepressant 2C18, 2F6Clozapine Neuroleptic 2D6 TiPS 1992, 13, 434-439 Codeine Analgesic 2D6 -demethylation Cyclosporin Immunospressant 3A4 hydroxylated and N-demethylated metabolites Dapsone Antibacterial 3A4 DebrisoquineAntihypertensive 2D6 Desipramine Antidepressant 2D6 TiPS 1992, 13,434-439 Dextromethorphan Antitussive 2D6 Diazepam CNS Depressant 2C18Diltiazam Antihypertensive 3A4 Ebastine Antihistamine 3A4, 2D6 Structuresimilar to terfenadine Encainide Antiarrhythmic 2D6 ErythromycinAntibiotic 3A4 3A, 1A2 noncompetitive inhibitor FelodipineAntihypertensive 3A4 Flecainde Antiarrhymic 2D6 FluoxetineAntidepressant 2D6, 3A? - 2D6 (S > R) J Pharm Exp Ther 1993, 266,Demethylation 964-971) Fluphenazine Neuroleptic 2D6 TiPS 1992, 13,434-439 Guanoxan Antihypertensive 2D6 TiPS 1992, 13, 434-439Hydrocortisone Antiinflammatory 3A4 Imipramine Antidepressant2D6-hydroxylation Biol Pharm Bull 1993, 16, 3A4, 2C19 - 571demethylation Indoramin Antihypertensive 2D6 TiPS 1992, 13, 434-439Ketoconazole Antifungal 3A4 > 1A2 > 2C, 2D6 Lidocane Anesthetic 3A4 ClinPharm Ther 1989, 46, 521-527 Loratadine Antihistamine 3A4, 2D6 formationof SCH 34117 Lovastatin Cholesterol 3A4 Arch Biochem Biophys 1991,lowing agent 290, 355 Mephenytoin Anticonvulsant 2C18 MetoprololAntihypertensive 2D6 Mexiletine Antiarrhythmic 2D6 TiPS 1992, 13,434-439 Nifedipine Antihypertensive 3A J Biol Chem 1986, 261, 5051-50601Nitrendipine Antihypertensive 3A4 Nortriptylin Antidepressant 2D6 TiPS1992, 13, 434-439 Omeprazole Antiulcer 3A4 (major), 2C18 2C18 ISSXProceedings Vol 3, 45-46; Inducer of 1A2, Clin Pharmacokin 1993, 25,450-482 Perphenazine Neurologic 2D6 TiPS 1992, 13, 434-439 PhenytoinAntiepileptic 3A, 2C18 Probable inducer of 3A4; Clin Pharmacokin 1993,25, 450-482 Propafenone Antiarrhythmic 3A4, 1A2 - Molec Pharm 1993, 43,120-126 demethylation, 2D6 Propanalol Antihypertensive 2C18, 2D6Quinidine Antiarrhymic 3A4 2D6 Ranitidine Antiulcer 2D6 Bunitrololantihypertensive 4-hydroxylation - 2D6

B. Tissue Sample Preparation

The source of target DNA for detecting mutations in biotransformationgenes is usually genomic. In adults, samples can conveniently beobtained from blood or mouthwash or cheek scraping epithelial cells.cDNA can be obtained only from tissues in which biotransformation genesare expressed. The liver is a good source, but a surgical biopsy isrequired to remove a sample from living patients.

C. Amplification

The target DNA is usually amplified by PCR. Primers can be readilydevised from the known genomic and cDNA sequences of biotransformationgenes. The selection of primers, of course, depends on the areas of thetarget sequence that are to be screened. The choice of primers alsodepends on the strand to be amplified. Because some nonallelic P450genes show a high degree of sequence identity, selection of primers canbe important in determining whether one or more nonallelic seqments isamplified. Usually, primers will be selected to be perfectlycomplementary to a unique sequence within a selected target resulting inamplification of only that target. Examples of suitable primers areshown in Table 6 (F=forward primer, R=reverse primer). TABLE 6 SEQUENCENAME SEQUENCE CYP2DE1F GCCAGGTGTGTCCAGAGGAGCCCAT CYP2DE1RCTGGTAGGGGAGCCTCAGCACCTCT CYP2DE2F TAGGACTAGGACCTGTAGTCTGGGGT CYP2DE2RGGTCCCACGGAAATCTGTCTCTGT CYP2DE34F CTAATGCCTTCATGGCCACGCGCA CYP2DE34RTCGGGAGCTCGCCCTGCAGAGA CYP2DE5F GGGCCTGAGACTTGTCCAGGTGAA CYP2DE5RCCCTCATTCCTCCTGGGACGCTCAA CYP2DE6F CCCGTTCTGTCCCGAGTATGCTCT CYP2DE6RTCGGCCCCTGCACTGTTTCCCAGA CYP2DE7F GCTGACCCATTGTGGGGACGCAT CYP2DE7RCTATCACCAGGTGCTGGTGCTGAGCT CYP2DE89F GGGAGACAAACCAGGACCTGCCAGA CYP2DE89RCTCAGCCTCAACGTACCCCTGTCT CYP2D678-F TGAGAGCAGCTTCAATGATGAGAACCTCYP2D678-R GTAGGATCATGAGCAGGAGGCCCCA CYP-PCR8-F TCCCCCGTGTGTTTGGTGGCACYP-PCR9-R TGCTTTATTGTACATTAGAGC

For analysis of mutants through all or much of a gene, it is oftendesirable to amplify several segments from several paired primers. Thedifferent segments may be amplified sequentially or simultaneously bymultiplex PCR. Frequently, fifteen or more segments of a gene aresimultaneously amplified by PCR. The primers and amplificationsconditions are preferably selected to generate fluorescently labelledDNA targets. Double stranded targets are enzymically degraded tofragments of about 100 bp and denatured before hybridization.

D. Tiling Strategies

Mutations in biotransformation genes can be detected by any of thetiling strategies noted above. For detection of hitherto uncharacterizedmutations, the basic tiling strategy is one suitable strategy. The chipscontain-probes tiling across some or all of a reference sequence.

For detecting precharacterized mutations, which account for the largemajority of poor metabolizers in the preferred reference genes describedabove, the block tiling strategy is one particularly useful approach. Inthis strategy, a group (or block) of probes is used to analyze a shortsegment of contiguous nucleotides (e.g., 3, 5, 7 or 9) from abiotransformation gene centered around the site of a mutation.

In a preferred embodiment, a first group of probes is tiled based on awildtype reference sequence and a second group is tiled based a mutantversion of the wildtype reference sequence. The mutation can be a pointmutation, insertion or deletion or any combination of these. Thepresence of first and second groups of probes facilitates analysis whenmultiple target sequences are simultaneously applied to the chip, as isthe case when a patient being diagnosed is heterozygous in abiotransformation gene. The principles of chip design and analysis areas described for the CFTR chip.

E. Modifications for Determining Gene Copy Number

As discussed in connection with the CFTR chip, the tiling arrays of theinvention are usually capable of simultaneously analyzing heterozygousalleles of a target sequence. The presence of heterozygous alleles issignalled by two probes having interrogations positions aligned with themutation showing specific hybridization, rather than one, as would bethe case for homozygous alleles. Interpretation of hybridizationpatterns is, however, sometimes complicated by the presence of lessthan, or more than, the two expected copies of a biotransformation genein an individual.

For example, an individual having one wildtype copy of the gene, and awholly deleted second copy of the gene would show a similarhybridization pattern to an individual with two wildtype copies (otherthan for possible differences in overall intensity of the pattern). Infact, complete gene deletions of one or both copies of a gene accountfor approximately 15% of slow metabolizers having defectivebiotransformation enzymes. Analogous loss of heterozygosity occurs inother diseases such as cancer (p53) and muscular dystrophy (dystrophingene).

Further, an individual with three wildtype copies of a biotransformationgene would show a similar hybridization pattern to an individual withtwo copies of the gene, other than for a difference in overallintensity. Individuals having multiple copies of a biotransformationgene are referred to as super metabolizers, because of their elevatedlevels of enzymes.

Additional complications in interpreting a hybridization pattern canresult from the presence of pseudogenes in an individual. A pseudogeneis an analog of a true gene that shows strong sequence identity to thetrue gene but is not expressed. Most pseudogenes having counterpartsamong the biotransformation genes have been sufficiently wellcharacterized that their presence can be avoided by appropriateselection of amplification primers (i.e., primers are selected thathybridize to the true gene of interest without hybridizing to thepseudogene). For example, 5′ TGA JAG CAG CTT CAA TGA TGA GAA CCT 3′ and5′ GTA GGA TCA TGA GCA GGA GGC CCC A 3′, can be used for amplifying exon6. However, occasionally a pseudogene might be unexpectedly amplifiedtogether with a true gene, and the presence of mutations in thepsuedogene (which in fact have no phenotypic effect) might be mistakenlythought to occur in the true gene.

The invention provides tiling arrays to overcome these difficulties byindicating how many copies of a target are present in a sample. Inaddition to containing the probes required for the tiling strategiesdescribed above, these arrays contain probes for analyzing polymorphicsites of a target gene, which do not exert any phenotypic effect (i.e.,silent polymorphic sites). The frequency and diversity of such sites isusually greater than that of mutations whose presence does exert aphenotypic effect. Silent sites are predominantly found in intronicregions and in flanking regions (i.e., within about 20 kb of transcribedregions), where selective pressure is generally lower relative to thecoding regions. Any number of additional polymorphic sites can be tiledusing the same strategies as previously described. For any particularpolymorphic site, each form of the polymorphism at that sites serves asa reference sequence for a separate tiling. In some instances, silentpolymorphic sites can be amplified from the same primers and on the sameamplicon as the sites of potential mutations. In other instances,separate amplification is required.

Silent polymorphic regions can be identified by comparing segments oftarget DNA, particularly introns and flanking regions, from differentindividuals. Comparison can be performed using the general tilingstrategies disclosed above or by conventional techniques such assingle-stranded conformational analysis. See, e.g., Ha yashi, PCRMethods & Applications 1, 34-38 (1991); Orita, Proc. Natl. Acad. Sci.USA 86, 2766-2270 (1989); Orita et al., Genomics 5, 874-879 (1989). Thismethod has been successfully employed in dystrophin gene analysiscoupled with heteroduplex formation to scan for new mutations. Prior etal., Human Molecular Genetics 2, 311-313 (1993).

Analysis of the hybridization pattern of a probe array tiling a silentpolymorphic region indicates which of the polymorphic forms are presentat this region. Consider a polymorphism constituting a single basechange. If the polymorphism and flanking sequences are tiled accordingto the basic strategy using four probe sets, there are four probeshaving an interrogation position aligned with the single base at whichthe polymorphism occurs. The number of these four probes to showspecific hybridization indicates the number of different polymorphicforms present, and hence, the minimum number of copies of a genepresent. For example, if two probes show specific hybridization, atleast two polymorphic forms are present. There may be more copies of thegene than polymorphic forms observed at any one site, because the samepolymorphic form may be present in more than one copy of the gene.However, if sufficient polymorphic sites are examined, it is likely thata site will be found at which each copy of the gene exists in adifferent polymorphic form. Thus, the copy number of a gene can bededuced from the number of polymorphic forms present at the polymorphicsite(s) showing the greatest number of polymorphic forms.

If a silent pqlymorphism is more complicated than a single-base change(e.g., deletion or insertion), the number of polymorphic forms can bedetermined from alternative tilings to the different forms, as generallydescribed in §I. B.1. For example, if all the perfectly matched probesin a first tiling hybridize, it is concluded that the polymorphic formconstituting the reference sequence for the first tiling is present. If,all the perfectly matched probes in two (or more) tilings hybridize, itis concluded that two (or more) polymorphic forms are present.

F. Applications

In general, the biotransformation genes described above are inherited inan autosomal recessive fashion. The presence of a homozygous mutation ortwo heterozygous mutations in an individual signals that the individualis a poor metabolizer of any drug metabolized by the biotransformationgene in which the mutation occurs. Some individuals with one mutant andone normal gene show a near wildtype phenotype, but other suchindividuals show an intermediate phenotype between normal and homozygousmutant. Individuals having additional copies of a biotransformation geneusually express the gene product at higher levels than a wildtypeindividual.

The screening methods can be routinely applied as precaution beforeadministering a drug to a patient for the first time. If the patient isfound to lack both copies of a gene expressing an enzyme required fordetoxification of a particular drug, the patient generally should not beadministered the drug or, should be administered the drug in smallerdoses compared with patients having normal levels of the enzyme. Thelatter course may be necessary if no alternative treatment is available.If the patient is found to lack both copies of a gene expressing anenzyme required for activation of a particular drug, the drug will haveno beneficial effect on the patient and should not be administered.Patients having one wildtype copy of a gene and one mutant copy of agene, and who are at risk of having lower levels of an enzyme, should beadministered drugs metabolized by that enzyme only with some caution,again depending on whether alternatives are available. If the drug isdetoxified by the enzyme in question, the patient should in general beadministered a lower dose of the drug. If the drug is activated by theenzyme, the heterozygous patient should be administered a higher dosageof the drug. The reverse applies for patients having additionalcopy(ies) of a particular biotransformation gene, who are at risk ofhaving elevated levels of an enzyme. The more rational selection oftherapeutic agents that can be made with the benefit of screeningresults in fewer side effects and greater drug efficacy in poormetabolizer patients.

The methods are also useful for screening populations of patients whoare to be used in a clinical trial of a new drug. The screeningidentifies a pool of patients, each of whom has wildtype levels of thefull complement of biotransformation enzymes. The pool of patients arethen used for determining safety and efficacy of the drugs. Drugs shownto be effective by such trials are formulated for therapeutic use with apharmaceutical carrier such as sterile distilled water, physiologicalsaline, Ringer's solution, dextrose solution, and Hank's solution.

The chips are also useful for screening patients for increased risk ofcancer in similar manner to the p53 chips of the invention. Somebiotransformation enzymes have roles in activating environmentalprocarcinogens to carcinogenic form (e.g., 1A1, 2D6, 2E1 andN-acetyltransferase). Mutations in genes encoding these enzymes areassociated with reduced cancer risk. Other biotransformation enzymeshave roles in detoxifying environmental carcinogens, e.g., glutathioneS-transferase M1. Mutations in one, and especially both, copies of genesencoding such enzymes are associated with enhanced susceptibility tocancer. See Shields, Environmental Health Perspectives 102 (sup. 11),81-87 (1994).

CYP genotype information can be useful to prevent drug-drug interactionsin two main ways. First, some drugs are known to inhibit specific CYPenzymes. When such a drug is given, care should be taken not to give asecond drug handled by the inhibited pathway (see Table 4). Second, whena person is genotyped as a poor metabolizer, not only should drug dosesbe decreased, second drugs handled by the poor metabolizing pathwayshould not be added to the therapy.

EXAMPLE

FIG. 10 shows the layout of probes and a computer-simulatedhybridization pattern for an exemplary chip containing tilings forCYP2D6 and CYP2C19 (wildtype). The chip contains a number of separatetilings as follows.

(1) A tiling (basic strategy) of all 9 exons plus 5 nucleotides of eachintron bordering the exons of the CYP2D6 gene. The probes were 14 merswith the interrogation position at nucleotide 7. This tiling is theupper right of the figure (excluding the eleven columns of probe sets onthe left of the chip). Each lane of probes is divided into four columns,occupied by probes differing at the interrogation position. At any onecolumn, a nucleotide in the target sequence aligned with the columnposition is identified as the complement of the nucleotide in the columnhaving the highest fluorescent intensity.

(2) A tiling (basic strategy) of the complete coding sequence(cDNA/mRNA) of CYP2C19 (wildtype). The probes were 15 mers with theinterrogation position at nucleotide 7. This tiling is in the lower halfof the figure (excluding the eleven columns of probes at the left of thefigure).

(3) A series of “opti-block” tilings for analysis of known mutations inCYP2D6 and CYP2C19. These blocks run down the left hand eleven columnsof the figure. These blocks are labelled 2C19 m2 (mutation in cytochromeP450 2C19), p34S, L91M, H94A, p1085, p1127, Delta T 1795, p1749, ss1934, G212E, 2637DeltaA, delta281, 296C, H324P, L421P, S486T, 2C19 ml(mutation in cytochrome P450 2C19). Unless otherwise indicated, themutations occur in cytochrome P450 2D6.

(4) A series of alternative tilings for analysis of known polymorphicdifferences between CYP2D6 and its pseudogenes CYP2D7P, CYP2D7AP andCYP2DAP. These tilings are also in the left hand column of the figure.These tilings are labelled Ex6p 2D6/2D7, Ex2p 2D6/2D7, Ex2p 2D6/2D8,Ex4p 2D6/2D7, Ex4p 2D6/2D8, Ex6p 2D6/2D8, Ex7p 2D6/2D7, 2D6/2D7.

FIG. 11 shows an alternative tiling designed to distinguish 2D6 from thepseudogene 2D7 in CYP2D6. Alternative tilings are formed from twointerdigitated tilings, each designed according to the basic tilingstrategy based on two different reference sequences, in this case 2D6and 2D7. The first column contains four probes complementary to theCYP2D6 sequence except at the interrogation position. The second columncontains four probes complementary to the CYP2D7 sequence except at theinterrogation position. The interrogation positions of the first andsecond columns of probes align with the same positions of the targetsequence. The same strategy of alternating probes from the respective2D6 and 2D7 reference sequences continues throughout the alternativetiling. When the tiling is hybridized to only the CYP2D6 form, onlyprobes complementary to CYP2D6 (i.e., the columns labelled 6) light up.Conversely when the tiling is hybridized to only the CYP2D7 form, onlyprobes in the columns labelled 7 light up. When the tiling is hybridizedto a mixture of CYP2D6 and CYP2D7, the pattern is the sum of the patternfor the two individual forms. The characteristic patterns throughout thetiling allow distinction of whether CYP2D6, CYP2D7 or both are present.

FIG. 12 shows an optiblock of probes for distinguishing the P34Smutation from the wildtype sequence of CYP2D6. In an optiblock, probesare selected based on the block tiling strategy. That is all probesalign with the same segment of target DNA but differ in the location ofthe interrogation position and in whether the probes are tiled based ona wildtype or mutant reference sequence. The notation “n” above the chipindicates that the interrogation position is aligned with the site ofthe P34S mutation in the target DNA and, the notation n−1 and n+1indication interrogation positions aligned one base either side of thesite of mutation, and so forth. As in the alternate tiling, probes tiledon wildtype and mutant sequences (sometimes referred to as wildtype andmutant probes) are interdigitated. The result of hybridizing theoptiblock to wildtype target is that all columns containing probes tiledbased on the wildtype sequence light up. In addition, one column ofprobes based on the mutant sequence lights up, this being the column ofprobes having an interrogation position aligned with the “n” nucleotidein the target. The result of hybridizing the optiblock to the mutanttarget is the reverse; that is all columns of probes tiled based on themutant target sequence light up, and a single column of probes tiledbased on the wildtype sequence lights up. When the optiblock ishybridized to a heterozygous target containing wildtype and mutantforms, the pattern is the sum of those obtained with the individualtargets alone. Thus, all three possible targets, homozygous wildtype,homozygous mutant and heterozygote give distinct patterns ofhybridization and dan be distinguished.

The chip was hybridized with fluorescein-labelled-dGTP double-strandedDNA made by PCR from a plasmid template containing the genomic clone ofCYP2D6-B. The entire gene is amplified as 4 separate PCR products, allof which were present during hybridization. dUTP was incorporated duringPCR and the PCR products were treated with uracil DNA glycosylase, thenheated to 95° C. for 5 min before hybridization to fragment and denaturedouble-stranded material. Hybridization was for 30 min at 37° C. in 0.5M LiCl plus 0.0005% NaLauroylSarkosine. Washing was performed prior toscanning the same solution without target DNA for 5 min at roomtemperature.

FIG. 13 shows the chip hybridized to a CYP2D6-B target. A portion of thebasic tiling pattern is shown magnified in the lower right hand corner.Successive nucleotides in the target sequence can be read by eye bycomparing the sequence intensities of the four squares in a column. Fromtop to bottom, these squares are respectively occupied by probes havingA, C, G and T at the interrogation position. The nucleotide occupyingthe position in the target sequence aligned with the interrogationposition of a column of probes is the complement of the interrogationposition of the probe showing the highest signal. The SS1934 :mutationin CYP2D6-B results in a G-A transition and loss of function. Theenlarged hybridization pattern in the lower right of the figure has anarrow in the column corresponding to nucleotide 1934. In this column,the probe hybridizing most strongly has a T in the interrogationposition. This implies that the corresponding nucleotide in the targetis the complement of T, i.e., A, indicating that the mutant form of thetarget is present. The same result is apparent from the optiblock shownin the upper left of the figure. This block shows three consecutivecolumns in which the T-probe lights up. Two of these columns are fromwildtype and mutant probes having interrogation positions aligned withnucleotide 1934. The third column (the leftmost of the three) is themutant probe having an interrogation position aligned with nucleotide1933.

FIG. 14 shows magnifications of the hybridization patterns of L421P andS486 opti-tiling blocks. In each case, the first, third, fifth, sixth,seventh, and ninth columns light up. This pattern indicates thathomozygous wildtype sequence is present (see the idealized pattern forhomozygous wildtype in FIG. 12).

In a separate experiment, the chip was hybridized to CYP2C19 cDNA, asshown in FIG. 15. The Figure shows that the lower-portion of the chipcontaining the 2C19 tiles is lit. A magnification of part of thehybridization pattern from the basic tiling sequence is shown in theupper right of the Figure. Again, the sequence can be read by eye bycomparing the intensities of the four probes forming a column.

III. Modes of Practicing The Invention

A. VLSIPS™ Technology

As noted above, the VLSIPS™ technology is described in a number ofpatent publications and is preferred for making the oligonucleotidearrays of the invention. A brief description of how this technology canbe used to make and screen DNA chips is provided in this Example and theaccompanying Figures. In the VLSIPS™ method, light is shone through amask to activate functional (for oligonucleotides, typically an —OH)groups protected with a photoremovable protecting group on a surface ofa solid support. After light activation, a nucleoside building block,itself protected with a photoremovable protecting group (at the 5′-OH),is coupled to the activated areas of the support. The process can berepeated, using different masks or mask orientations and buildingblocks, to prepare very dense arrays of many different oligonucleotideprobes. The process is illustrated in FIG. 16; FIG. 17 illustrates howthe process can be used to prepare “nucleoside combinatorials” oroligonucleotides synthesized by coupling all four nucleosides to formdimers, trimers and so forth.

New methods for the combinatorial chemical synthesis of peptide,polycarbamate, and oligonucleotide arrays have recently been reported(see Fodor et al., 1991, Science 251: 767-773; Cho et al., 1993, Science261: 1303-1305; and. Southern et al., 1992, Genomics 13: 1008-10017,each of which is incorporated herein by reference). These arrays, orbiological chips (see Fodor et al., 1993, Nature 364: 555-556,incorporated herein by reference), harbor specific chemical compounds atprecise locations in a high-density, information rich format, and are apowerful tool for the study of biological recognition processes. Aparticularly exciting application of the array technology is in thefield of DNA sequence analysis. The hybridization pattern of a DNAtarget to an array of shorter oligonucleotide probes is used to gainprimary structure information of the DNA target. This format hasimportant applications in sequencing by hybridization, DNA diagnosticsand in elucidating the thermodynamic parameters affecting nucleic acidrecognition.

Conventional DNA sequencing technology is a laborious procedurerequiring electrophoretic size separation of labeled DNA fragments. Analternative approach, termed Sequencing By Hybridization (SBH), has beenproposed (Lysov et al., 1988, Dokl.Akad.Nauk SSSR 303:1508-1511; Bainset al., 1988, J. Theor. Biol. 135:303-307; and Drmanac et al., 1989,Genomics 4:114-128, incorporated herein by reference and discussed inDescription of Related Art, supra). This method uses a set of shortoligonucleotide probes of defined sequence to search for complementarysequences on a longer target strand of DNA. The hybridization pattern isused to reconstruct the target DNA sequence. It is envisioned thathybridization analysis of large numbers of probes can be used tosequence long stretches of DNA. In immediate applications of thismethodology, a small number of probes can be used to interrogate localDNA sequence. The strategy of SBH can be illustrated by the followingexample. A 12-mer target DNA sequence, AGCCTAGCTGAA, is mixed with acomplete set of octanucleotide probes. If only perfect complementarityis considered, five of the 65,536 octamer probes -TCGGATCG, CGGATCGA,GGATCGAC, GATCGACT, and ATCGACTT will hybridize to the target. Alignmentof the overlapping sequences from the hybridizing probes reconstructsthe complement of the original 12-mer target: TCGGATCG  CGGATCGA  GGATCGAC    GATCGACT     ATCGACTT TCGGATCGACTT

Hybridization methodology can be carried out by attaching target DNA toa surface. The target is interrogated with a set of oligonucleotideprobes, one at a time (see Strezoska et al., 1991, Proc. Natl. Acad.Sci. USA 88:10089-10093, and Drmanac et al., 1993, Science260:1649-1652, each of which is incorporated herein by reference). Thisapproach can be implemented with well established methods ofimmobilization and hybridization detection, but involves a large numberof manipulations. For example, to probe a sequence utilizing a full setof octanucleotides, tens of thousands of hybridization reactions must beperformed. Alternatively, SBH can be carried out by attaching probes toa surface in an array format where the identity of the probes at eachsite is known. The target DNA is then added to the array of probes. Thehybridization pattern determined in a single experiment directly revealsthe identity of all complementary probes.

As noted above, a preferred method of oligonucleotide probe arraysynthesis involves the use of light to direct the synthesis ofoligonucleotide probes in high-density, miniaturized arrays. Photolabile5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linkerchemistry, and versatile combinatorial synthesis strategies have beendeveloped for this technology. Matrices of spatially-definedoligonucleotide probes have been generated, and the ability to use thesearrays to identify complementary sequences has been demonstrated byhybridizing fluorescent labeled oligonucleotides to the DNA chipsproduced by the methods. The hybridization pattern demonstrates a highdegree of base specificity and reveals the sequence of oligonucleotidetargets.

The basic strategy for light-directed oligonucleotide synthesis (1) isoutlined in FIG. 18. The surface of a solid support modified withphotolabile protecting groups (X) is illuminated through aphotolithographic mask, yielding reactive hydroxyl groups in theilluminated regions. A 3′-O-phosphoramidite activated deoxynucleoside(protected at the 5′-hydroxyl with a photolabile group) is thenpresented to he surface and coupling occurs at sites that were exposedto light. Following capping, and oxidation, the substrate is rinsed andthe surface illuminated through a second mask, to expose additionalhydroxyl groups for coupling. A second 5-protected, 3′-O-phosphoramiditeactivated deoxynucleoside is presented to the surface. The selectivephotodeprotection and coupling cycles are repeated until the desired setof products is obtained.

Light directed chemical synthesis lends itself to highly efficientsynthesis strategies which will generate a maximum number of compoundsin a minimum number of chemical steps. For example, the complete set of₄ ^(n) polynucleotides (length n), or any subset of this set can beproduced in only 4×n chemical steps. See FIG. 17. The patterns ofillumination and the order of chemical reactants ultimately define theproducts and their locations. Because photolithography is used, theprocess can be miniaturized to generate high-density arrays ofoligpnucleotide probes. For an example of the nomenclature useful fordescribing such arrays, an array containing all possible octanucleotidesof dA and dT is written as (A+T)⁸. Expansion of this polynomial revealsthe identity of all 256 octanucleotide probes from AAAAAAAA to TTTTTTTT.A DNA array composed of complete sets of dinucleotides is referred to ashaving a complexity of 2. The array given by (A+T+C+G)8 is the full65,536 octanucleotide array of complexity four. Computer-aided methodsof laying down predesigned arrays of probes using VLSIPS™ technology aredescribed in commonly-assigned co-pending application U.S. Ser. No.08/249,188, filed May 24, 1994 (incorporated by reference in itsentirety for all purposes).

In a variation of the VLSIPS™ methods, multiple copies of an array ofprobes are synthesized simultaneously. The multiple copies areeffectively stacked in a pile during the synthesis process in a mannersuch that each copy is accessible to irradiation. For example, synthesiscan occur through the volume of a slab of polymer gel that istransparent to the source of radiation used to remove photoprotectivegroups. Suitable polymers are described in U.S. Ser. No. 08/431,196,filed Apr. 27, 1995 (incorporated by reference in its entirety for allpurposes). For example, a polymer formed from a 90:10% w/w mixture ofacrylamide and N-2-aminoethylacrylamide is suitable.

After synthesis, the gel is sliced into thin layers (e.g., with amicrotome). Each layer is attached to a glass substrate to constitute aseparate chip. Alternatively, a pile can be formed from layers of gelseparated by layers of a transparent substance that can be mechanicallyor chemically removed after synthesis has occurred. Using these methods,up to about 10, 100 or 1000 identical arrays can be synthesizedsimultaneously.

To carry out hybridization of DNA targets to the probe arrays, thearrays are mounted in a thermostatically controlled hybridizationchamber. Fluorescein labeled DNA targets are injected into the chamberand hybridization is allowed to proceed for 5 min to 24 hr. The surfaceof the matrix is scanned in an epifluorescence microscope (ZeissAxioscop 20) equipped with photon counting electronics using 50-100 μWof 488 nm excitation from an Argon ion laser (Spectra Physics Model2020). Measurements may be made with the target solution in contact withthe probe matrix or after washing. Photon counts are stored and imagefiles are presented after conversion to an eight bit image format. SeeFIG. 21.

When hybridizing a DNA target to an oligonucleotide array, N=Lt−(Lp−1)complementary hybrids are expected, where N is the number of hybrids, Ltis the length of the DNA target, and Lp is the length of theoligonucleotide probes on the array. For example, for an 11-mer targethybridized to an octanucleotide array, N=4. Hybridizations withmismatches at positions that are 2 to 3 residues from either end of theprobes will generate detectable signals. Modifying the above expressionfor N, one arrives at a relationship estimating the number of detectablehybridizations (Nd) for a DNA target of length Lt and an array ofcomplexity C. Assuming an average of 5 positions giving signals abovebackground:

-   -   Nd=(1+5(C−1)) [Lt−(Lp−1)].

Arrays of oligonucleotides can be efficiently generated bylight-directed synthesis and can be used to determine the identity ofDNA target sequences. Because combinatorial strategies are used, thenumber of compounds increases exponentially while the number of chemicalcoupling cycles increases only linearly. For example, synthesizing thecomplete set of 48 (65,536) octanucleotides will add only four hours tothe synthesis for the 16 additional cycles. Furthermore, combinatorialsynthesis strategies can be implemented to generate arrays of anydesired composition. For example, because the entire set of dodecamers(4¹²) can be produced in 4⁸ photolysis and coupling cycles (b^(n)compounds requires b×n cycles), any subset of the dodecamers (includingany subset of shorter oligonucleotides) can be constructed with thecorrect lithographic mask design in 48 or fewer chemical coupling steps.In addition, the number of compounds in an array is limited only by thedensity of synthesis sites and the overall array size. Recentexperiments have demonstrated hybridization to probes synthesized in 25μm sites. At this resolution, the entire set of 65,536 octanucleotidescan be placed in an array measuring 0.64 cm square, and the set of1,048,576 dodecanucleotides requires only a 2.56 cm array.

Genome sequencing projects will ultimately be limited by DNA sequencingtechnologies. Current sequencing methodologies are highly reliant oncomplex procedures and require substantial manual effort. Sequencing byhybridization has the potential for transforming many of the manualefforts into more efficient and automated formats. Light-directedsynthesis is an efficient means for large scale production ofminiaturized arrays for SBH. The oligonucleotide arrays are not limitedto primary sequencing applications. Because single base changes causemultiple changes in the hybridization pattern, the oligonucleotidearrays provide a powerful means to check the accuracy of previouslyelucidated DNA sequence, or to scan for changes within a sequence. Inthe case of octanucleotides, a single base change in the target DNAresults in the loss of eight complements, and generates eight newcomplements. Matching of hybridization patterns may be useful inresolving sequencing ambiguities from standard gel techniques, or forrapidly detecting DNA mutational events. The potentially very highinformation content of light-directed oligonucleotide arrays will changegenetic diagnostic testing. Sequence comparisons of hundreds tothousands of different genes will be assayed simultaneously instead ofthe current one, or few at a time format. Custom arrays can also beconstructed to contain genetic markers for the rapid identification of awide variety of pathogenic organisms.

Oligonucleotide arrays can also be applied to study the sequencespecificity of RNA or protein-DNA interactions. Experiments can bedesigned to elucidate specificity rules of non Watson-Crickoligonucleotide structures or to investigate the use of novel syntheticnucleoside analogs for antisense or triple helix applications. Suitablyprotected RNA monomers may be employed for RNA synthesis. Theoligonucleotide arrays should find broad application deducing thethermodynamic and kinetic rules governing formation and stability ofoligonucleotide complexes.

Other than the use of photoremovable protecting groups, the nucleosidecoupling chemistry is very similar to that used routinely today foroligonucleotide synthesis. FIG. 18 shows the deprotection, coupling, andoxidation steps of a solid phase DNA synthesis method. FIG. 19 shows anillustrative synthesis route for the nucleoside building blocks used inthe method. FIG. 20 shows a preferred photoremovable protecting group,MeNPOC, and how to prepare the group in active form. The proceduresdescribed below show how to prepare these reagents. The nucleosidebuilding blocks are 5′-MeNPOC-THYMIDINE-3′-OCEP; 5′-MeNPOC-N⁴-t-BUTYLPHENOXYACETYL-DEOXYCYTIDINE-3′-OCEP; 5′-MeNPOC-N⁴-t-BUTYLPHENOXYACETYL-DEOXYGUANOSINE-3′-OCEP; and 5′-MeNPOC-N⁴-t-BUTYLPHENOXYACETYL-DEOXYADENOSINE-3′-OCEP.1. Preparation of 4,5-methylenedioxy-2-nitroacetophenone

A solution of 50 g (0.305 mole) 3,4-methylenedioxy-acetophenone(Aldrich) in 200 mL glacial acetic acid was added dropwise over 30minutes to 700 mL of cold (2-4° C.) 70% HNO₃ with stirring (NOTE: thereaction will overheat without external cooling from an ice bath, whichcan be dangerous and lead to side products). At temperatures below 0°C., however, the reaction can be sluggish. A temperature of 3-5° C.seems to be optimal). The mixture was left stirring for another 60minutes at 3-5° C., and then allowed to approach ambient temperature.Analysis by TLC (25% EtOAc in hexane) indicated complete conversion ofthe starting material within 1-2 hr. When the reaction was complete, themixture was poured into ^(˜)3 liters of crushed ice, and the resultingyellow solid was filtered off, washed with water and then suction-dried.Yield ^(˜)53 g (84%), used without further purification.2. Preparation of 1-(4,5-Methylenedioxy2-nitrophenyl) ethanol

Sodium borohydride (10 g; 0.27 mol) was added slowly to a cold, stirringsuspension of 53 g (0.25 mol) of 4,5-methylenedioxy-2-nitroacetophenonein 400 mL methanol. The temperature was kept below 10° C. by slowaddition of the NaBH₄ and external cooling with an ice bath. Stirringwas continued at ambient temperature for another two hours, at whichtime TLC (CH₂Cl₂) indicated complete conversion of the ketone. Themixture was poured into one liter of ice-water and the resultingsuspension was neutralized with ammonium chloride and then extractedthree times with 400 mL CH₂Cl₂ or EtOAc (the product can be collected byfiltration and washed at this point, but it is somewhat soluble in waterand this results in a yield of only ^(˜)60%). The combined organicextracts were washed with brine, then dried with MgSO₄ and evaporated.The crude product was purified from the main byproduct by dissolving itin a minimum volume of CH₂Cl₂ or THF(^(˜)175 ml) and then precipitatingit by slowly adding hexane (1000 ml) while stirring (yield 51 g; 80%overall). It can also be recrystallized (e.g., toluene-hexane), but thisreduces the yield.3. Preparation of 1-(4,5-methylenedioxy-2-nitrophenyl) ethylchloroformate (MeNPOC-Cl)

Phosgene (500 mL of 20% w/v in toluene from Fluka: 965 mmole; 4 eq.) wasadded slowly to a cold, stirring solution of. 50g (237 mmole; 1 eq.) of1-(4,5-methylenedioxy-2-nitrophenyl) ethanol in 400 mL dry The solutionwas stirred overnight at ambient temperature at which point TLC (20%Et₂O/hexane) indicated >95% conversion. The mixture was evaporated (anoil-less pump with downstream aqueous NaOH trap is recommended to removethe excess phosgene) to afford a viscous brown oil. Purification waseffected by flash chromatography on a short (9×13 cm) column of silicagel eluted with 20% Et₂O/hexane. Typically 55 g (85%) of the solidyellow MeNPOC-Cl is obtained by this procedure. The crude material hasalso been recrystallized in 2-3 crops from 1:1 ether/hexane. On thisscale, ^(˜)100 ml is used for the first crop, with a few percent THFadded to aid dissolution, and then cooling overnight at −20° C. (thisprocedure has not been optimized). The product should be storeddesiccated at −20° C.

4. Synthesis of 5′-Menpoc-2′-deoxynucleoside-3′- (N,N-diisopropyl2-cyanoethyl phosphoramidites

(a) 5′-MeNPOC-Nucleosides

Base=THYMIDINE (T); N-4-ISOBUTYRYL 2′-DEOXYCYTIDINE (ibu-dC);N-2-PHENOXYACETYL 2′DEOXYGUANOSINE (PAC-dG); and N-6-PHENOXYACETYL2′DEOXYADENOSINE (PAC-dA)

All four of the 5′-MeNPOC nucleosides were prepared from thebase-protected 2′-deoxynucleosides by the following procedure. Theprotected 2′-deoxynucleoside (90 mmole) was dried by co-evaporatingtwice with 250 mL anhydrous pyridine. The nucleoside was then dissolvedin 300 mL anhydrous pyridine (or 1:1 pyridine/DMF, for the dGPACnucleoside) under argon and cooled to ^(˜)2° C. in an ice bath. Asolution of 24.6 g (90 mmole) MeNPOC-Cl in 100 mL dry THF was then addedwith stirring over 30 minutes. The ice bath was removed, and thesolution allowed to stir overnight at room temperature (TLC: 5-10% MeOHin CH₂Cl₂; two diastereomers). After evaporating the solvents undervacuum, the crude material was taken up in 250 mL ethyl acetate andextracted with saturated aqueous NaHCO₃ and brine. The organic phase wasthen dried over Na₂SO₄, filtered and evaporated to obtain a yellow foam.The crude products were finally purified by flash chromatography (9×30cm silica gel column eluted with a stepped gradient of 2% - 6% MeOH inCH₂Cl₂). Yields of the purified diastereomeric mixtures are in the rangeof 65-75%.(b) 5′-Menpoc-2′-deoxynucleoside-3′-(N,N- diisopropyl 2-cyanoethylphosphoramidites)

The four deoxynucleosides were phosphitylated using either 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite, or 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite. The following is a typicalprocedure. Add 16.6g (17.4 ml; 55 mmole) of 2-cyanoethyl-N, N,N′,N′-tetraisopropylphosphoro-diamidite to a solution of 50 mmole5′-MeNPOC-nucleoside and 4.3 g (25 mmole) diisopropylammoniumtetrazolide in 250 mL dry CH₂Cl₂ under argon at ambient temperature.Continue stirring for 4-16 hours (reaction monitored by TLC: 45:45:10hexane/CH₂Cl₂/Et₃N). Wash the organic phase with saturated aqueousNaHCO₃ and brine., then dry over Na₂SO₄, and evaporate to dryness.Purify the crude amidite by flash chromatography (9×25 cm silica gelcolumn eluted with hexane/CH₂Cl₂/TEA-45:45:10 for A, C, T; or 0:90:10for G). The yield of purified amidite is about 90% .

B. Preparation of Labeled DNA/Hybridization To Array

1. PCR

PCR amplification reactions are typically conducted in a mixturecomposed of, per reaction: 1 μl genomic DNA; 10 μl each primer (10pmol/μl stocks); 10 μl 10× PCR buffer (100 mM Tris.Cl pH8.5, 500 mM KCl,15 mM MgCl₂); 10 μl 2 mM dNTPs (made from 100 mM dNTP stocks); 2.5 U Taqpolymerase (Perkin Elmer AmpliTaq™, 5 U/μl); and H₂O to 100 μl . Thecycling conditions are usually 40 cycles (94° C. 45 sec, 55° C. 30 sec,72° C. 60 sec) but may need to be varied considerably from sample typeto sample type. These conditions are for 0.2 mL thin wall tubes in aPerkin Elmer 9600 thermocycler. See Perkin Elmer 1992/93 catalogue for9600 cycle time information. Target, primer length and sequencecomposition, among other factors, may also affect parameters.

For products in the 200 to 1000 bp size range, check 2 μl of thereaction on a 1.5% 0.5× TBE a garose gel using an appropriate sizestandard (phiX174 cut with HaeIII is convenient). The PCR reactionshould yield several picomoles of product. It is helpful to include anegative control (i.e., 1 μl TE instead of genomic DNA) to check forpossible contamination. To avoid contamination, keep PCR products fromprevious experiments away from later reactions, using filter tips asappropriate. Using a set of working solutions and storing mastersolutions separately is helpful, so long as one does not contaminate themaster stock solutions.

For simple amplifications of short fragments from genomic DNA it is, ingeneral, unnecessary to optimize Mg²⁺ concentrations. A good procedureis the following: make a master mix minus enzyme; dispense the genomicDNA samples to individual tubes or reaction wells; add enzyme to themaster mix; and mix and dispense the master solution to each well, usinga new filter tip each time..

2. Purification

Removal of unincorporated nucleotides and primers from PCR samples canbe accomplished using the Promega Magic PCR Preps DNA purification kit.One can purify the whole sample, following the instructions suppliedwith the kit (proceed from section IIIB, Sample preparation for directpurification from PCR reactions’). After elution of the PCR product in50 μl of TE or H₂O, one centrifuges the eluate for 20 sec at 12,000 rpmin a microfuge and carefully transfers 45 μl to a new microfuge tube,avoiding any visible pellet. Resin is sometimes carried over during theelution step. This transfer prevents accidental contamination of thelinear amplification reaction with ‘Magic PCR’ resin. Other methods,e.g., size exclusion chromatography, may also be used.

3. Linear Amplification

In a 0.2 mL thin-wall PCR tube mix: 4 μl purified PCR product; 2 μlprimer (10 pmol/μl); 4 μl 10× PCR buffer; 4 μl dNTPs (2 mM dA, dC, dG,0.1 mM dT); 4 μl 0.1 mM dUTP; 1 μl 1 mM fluorescein dUTP (Amersham RPN2121); 1 U Taq polymerase (Perkin Elmer, 5 U/μl); and add H2O to 40 μl.Conduct 40 cycles (92° C. 30 sec, 55° C. 30 sec, 72° C. 90 sec) of PCR.These conditions have been used to amplify a 300 nucleotidemitochondrial DNA fragment but are applicable to other fragments. Evenin the absence of a visible product band on an agarose gel, there shouldstill be enough product to give an easily detectable hybridizationsignal. If one is not treating the DNA with uracil DNA glycosylase (seeSection 4), dUTP can be omitted from the reaction.

4. Fragmentation

Purify the linear amplification product using the Promega Magic PCRPreps DNA purification kit, as per Section 2 above. In a 0.2 mLthin-wall PCR tube mix: 40 μl purified Labeled DNA; 4 μl 10× PCR buffer;and 0.5 μl uracil DNA glycosylase (BRL 1U/μl). Incubate the mixture 15min at 37° C., then 10 min at 97° C.; store at −20° C. until ready touse.

5. Hybridization, Scanning & Stripping

A blank scan of the slide in hybridization buffer only is helpful tocheck that the slide is ready for use. The buffer is removed from theflow cell and replaced with 1 ml of (fragmented) DNA in hybridizationbuffer and mixed well optionally, standard hybridization buffer can besupplemented with tetramethylammonium chloride (TMACL) or betaine(N,N,N-trimethylglycine; (CH₃)₃ N+CH₂COO⁻) to improve discriminationbetween perfectly matched targets and single-base mismatches. Betaine iszwitterionic at neutral pH and alters the composition-dependentstability of nucleic acids without altering their polyelectrolytebehavior. Betaine is preferably used at a concentration between 1 and 10M and, optimally, at about 5 M. For example, 5 M betaine in 2×SSPE issuitable. Inclusion of betaine at this concentration lowers the averagehybridization signal about four fold, but increases the discriminationbetween matched and mismatched probes.

The scan is performed in the presence of the labeled target. FIG. 21illustrates an illustrative detection system for scanning a DNA chip. Aseries of scans at 30 min intervals using a hybridization temperature of25° C. yields a very clear signal, usually in at least 30 min to twohours, but it may be desirable to hybridize longer, i.e., overnight.Using a laser power of 50 μW and 50 μm pixels, one should obtain maximumcounts in the range of hundreds to low thousands/pixel for a new slide.When finished, the slide can be stripped using 50% formamide. rinsingwell in deionized H₂O, blowing dry, and storing at room temperature.

C. Preparation of Labeled RNA/Hybridization To Array

1. Tagged Primers

The primers used to amplify the target nucleic acid should have promotersequences if one desires to produce RNA from the amplified nucleic acid.Suitable promoter sequences are shown below and include: (1) the T3promoter sequence: 5′-CGGAATTAACCCTCACTAAAGG 5′-AATTAACCCTCACTAAAGGGAG;(2) the T7 promoter sequence: 5′-TAATACGACTCACTATAGGGAG; and (3) the SP6promoter sequence: 5′ ATTTAGGTGACACTATAGAA.

The desired promoter sequence is added to the 5′ end of the PCR primer.It is convenient to add a different promoter to each primer of a PCRprimer pair so that either strand may be transcribed from a single PCRproduct.

Synthesize PCR primers so as to leave the DMT group on. DMT-onpurification is unnecessary for PCR but appears to be important fortranscription. Add 25 μl 0.5 M NaOH to collection vial prior tocollection of oligonucleotide to keep the DMT group on. Deprotect usingstandard chemistry—−55° C. overnight is convenient.

HPLC purification is accomplished by drying down the oligonucleotides,resuspending in 1 mL 0.1 M TEAA (dilute 2.0 M stock in deionized water,filter through 0.2 micron filter) and filter through 0.2 micron filter.Load 0.5 mL on reverse phase HPLC (column can be a Hamilton PRP-1semi-prep, #79426). The gradient is 0→50% CH₃CN over 25 min (program 0.2μmol.prep.0-50, 25 min). Pool the desired fractions, dry down, resuspendin 200 μl 80% HAc. 30 min RT. Add 200 μl EtOH; dry down. Resuspend in200 μl H₂O, plus 20 μl NaAc pH5.5, 600 μl EtOH. Leave 10 min on ice;centrifuge 12,000 rpm for 10 min in microfuge. Pour off supernatant.Rinse pellet with 1 mL EtOH, dry, resuspend in 200 μl H2O. Dry,resuspend in 200 μl TE. Measure A260, prepare a 10 pmol/μl solution inTE (10 mM Tris.Cl pH 8.0, 0.1 mM EDTA). Following HPLC purification of a42 mer, a yield in the vicinity of 15 nmol from a 0.2 μmol scalesynthesis is typical.

2. Genomic DNA Preparation

Add 500 μl (10 mM Tris.Cl pH8.0, 10 mM EDTA, 100 mM NaCl, 2% (w/v) SDS,40 mM DTT, filter sterilized) to the sample. Add 1.25 μl 20 mg/mlproteinase K (Boehringer) Incubate at 55° C. for 2 hours, vortexing onceor twice. Perform 2×0.5 mL 1:1 phenol:CHCl₃ extractions. After eachextraction, centrifuge 12,000 rpm 5 min in a microfuge and recover 0.4mL supernatant. Add 35 μl NaAc pH5.2 plus 1 mL EtOH. Place sample on ice45 min; then centrifuge 12,000 rpm 30 min, rinse, air dry 30 min, andresuspend in 100 μl TE.

3. PCR

PCR is performed in a mixture containing, per reaction: 1 μl genomicDNA; 4 μl each primer (10 pmol/μl stocks); 4 μl 10× PCR buffer (100 mMTris.Cl pH8.5, 500 mM KCl, 15 mM MgCl₂); 4 μl 2 mM dNTPs (made from 100mM dNTP stocks); 1 U Taq polymerase (Perkin Elmer,. 5 U/μl); H₂O to 40μl . About 40 cycles (94° C. 30 sec, 55° C. 30 sec, 72° C 30 sec) areperformed, but cycling conditions may need to be varied. Theseconditions are for 0.2 mL thin wall tubes in Perkin Elmer 9600. Forproducts in the 200 to 1000 bp size range, check 2 μl of the reaction ona 1.5% 0.5×TBE agarose gel using an appropriate size standard. Forlarger or smaller volumes (20-100 μl), one can use the same amount ofgenomic DNA but adjust the other ingredients accordingly.

4. In Vitro Transcription

Mix: 3 μl PCR product; 4 μl 5×buffer; 2 μl DTT; 2.4 μl 10 mM rNTPs (100mM solutions from Pharmacia); 0.48 μl 10 mM fluorescein-UTP(Fluorescein-12-UTP, 10 mM solution, from Boehringer Mannheim); 0.5 μlRNA polymerase (Promega T3 or T7 RNA polymerase); and add H₂O to 20 μl.Incubate at 37° C. for 3 h. Check 2 μl of the reaction on a 1.5% 0.5×TBEagarose gel using a size standard. 5×buffer is 200 mM Tris pH. 7.5, 30MM MgCl₂, 10 mM spermidine, 50 mM NaCl, and 100 mM DTT (supplied withenzyme). The PCR product needs no purification and can be added directlyto the transcription mixture. A 20 μl reaction is suggested for aninitial test experiment and hybridization; a 100 μl reaction isconsidered “preparative” scale (the reaction can be scaled up to obtainmore target) The amount of PCR product to add is variable; typically aPCR reaction will yield several picomoles of DNA. If the PCR reactiondoes not produce that much target, then one should increase the amountof DNA added to the transcription reaction (as well as optimize thePCR). The ratio of fluorescein-UTP to UTP suggested above is 1:5, butratios from 1:3 to 1:10—z,999 work well. One can also label withbiotin-UTP and detect with streptavidin-FITC to obtain similar resultsas with fluorescein-UTP detection.

For nondenaturing agarose gel electrophoresis of RNA, note that the RNAband will normally migrate somewhat faster than the DNA template band,although sometimes the two bands will comigrate. The temperature of thegel can effect the migration of the RNA band. The RNA produced from invitro transcription is quite stable and can be stored for months (atleast) at −20° C. without any evidence of degradation. It can be storedin unsterilized 6×SSPE 0.1% triton X-100 at −20° C. for days (at least)and reused twice (at least) for hybridization, without taking anyspecial precautions in preparation or during use. RNase contaminationshould of course be avoided. When extracting RNA from cells, it ispreferable to work very rapidly and to use strongly denaturingconditions. Avoid using glassware previously contaminated with RNases.Use of new disposable plasticware (not necessarily sterilized) ispreferred, as new plastic tubes, tips, etc., are essentially RNase free.Treatment with DEPC or autoclaving is typically not necessary.

5. Fragmentation

Heat transcription mixture at 94 degrees for forty min. The extent offragmentation is controlled by varying Mg²⁺ concentration (30 mM istypical), temperature, and duration of heating.

6. Hybridization, Scanning & Stripping

A blank scan of the slide in hybridization buffer only is helpful tocheck that the slide is ready for use. The buffer is removed from theflow cell and replaced with 1 mL of (hydrolysed) RNA in hybridizationbuffer and mixed well. Incubate for 15-30 min at 18° C. Remove thehybridization. solution, which can be saved for subsequent experiments.Rinse the flow cell 4-5 times with fresh changes of 6×SSPE 0.1% TritonX-100, equilibrated to 18° C. The rinses can be performed rapidly, butit is important to empty the flow cell before each new rinse and to mixthe liquid in the cell thoroughly. A series of scans at 30 min intervalsusing a hybridization temperature of 25° C. yields a very clear signal,usually in at least 30 min to two hours, but it may be desirable tohybridize longer, i.e., overnight. Using a laser power of 50 μW and 50μm pixels, one should obtain maximum counts in the range of hundreds tolow thousands/pixel for a new slide. When finished, the slide can bestripped using warm water.

These conditions are illustrative and assume a probe length of ^(˜)15nucleotides. The stripping conditions suggested are fairly severe, butsome signal may remain on the slide if the washing is not stringent.Nevertheless, the counts remaining after the wash should be very low incomparison to the signal in presence of target RNA. In some cases, muchgentler stripping conditions are effective. The lower the hybridizationtemperature and the longer the duration of hybridization, the moredifficult it is to strip the slide. Longer targets may be more difficultto -strip than shorter targets.

7. Amplification of Signal

A variety of methods can be used to enhance detection of labelledtargets bound to a probe on the array. In one embodiment, the proteinMutS (from E. coli) or equivalent proteins such as yeast MSH1, MSH2, andMSH3; mouse Rep-3, and Streptococcus Hex-A, is used in conjunction withtarget hybridization to detect probe-target complex that containmismatched base pairs. The protein, labeled directly or indirectly, canbe added to the chip during or after hybridization of target nucleicacid, and differentially binds to homo- and heteroduplex nucleic acid. Awide variety of dyes and other labels can be used for similar purposes.For instance, the dye YOYO-1 is known to bind preferentially to nucleicacids containing sequences comprising runs of 3 or more G residues.

8. Detection Of Repeat Sequences

In some circumstances, i.e., target nucleic acids with repeatedsequences or with high G/C content, very long probes are sometimesrequired for optimal detection. In one embodiment for detecting specificsequences in a target nucleic acid with a DNA chip, repeat sequences aredetected as follows. The chip comprises probes of length sufficient toextend into the repeat region varying distances from each end. Thesample, prior to hybridization, is treated with a labelledoligonucleotide that is complementary to a repeat region but shorterthan the full length of the repeat. The target nucleic is labelled witha second, distinct label. After hybridization, the chip is scanned forprobes that have bound both the labelled target and the labelledoligonucleotide probe; the presence of such bound probes shows that atleast two repeat sequences are present..

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1. An array comprising one or more array information features.
 2. Thearray of claim 1, wherein said one or more array information featurescomprises at least 4 features.
 3. The array of claim 2, wherein saidfeatures are positioned in a defined pattern on said array.
 4. The arrayof claim 3, wherein said defined pattern provides a symbol whenspecifically bound to target.
 5. The array of claim 1, wherein said oneor more features provides coded information when specifically bound totarget.
 6. The array of claim 5, wherein said coded information isbinary or non-binary coded information.
 7. A method for providinginformation about an array, said method comprising: contacting an arrayof claim 1 with a sample comprising a target that binds to at least oneof said one or more information features to produce at least one signalthat provides information about said microarray.
 8. The method of claim7, wherein said target is spiked into said sample prior to contacting ofsaid array with said sample.
 9. The method of claim 7, wherein saidinformation is provided by assessing binding of said target to said oneor more array information probes.
 10. The method of claim 9, whereinsaid assessing is by determining the presence, absence or level ofbinding to control levels of binding.
 11. The method of claim 7, furthercomprising determining the presence, absence or level of at least onesignal that provides said information.
 12. The method of claim 11,wherein said at least one signal provides a binary code, where 0 isrepresented by no detectable signal and 1 is represented by a detectablesignal.
 13. The method of claim 11, wherein said at least one signalprovides a binary code, where 1 is represented by no detectable signaland 0 is represented by a detectable signal.
 14. The method of claim 11,wherein said at least one signal provides a binary code, where 0 isrepresented by a signal generated by a first label and 1 is representedby a signal generated by a second label that is detectablydistinguishable from the first label.
 15. The method of claim 7, furthercomprising determining a level of said at least one signal to provide anon-binary code that provides said information.
 16. The method of claim15, wherein said non-binary code is represented by levels of signalrelative to a control level of signal.
 17. A composition comprising alabeled array information target that specifically binds to an arrayinformation probe.
 18. A kit comprising: (a) an array information probe;and (b) a target that binds to said array information probe underspecific binding conditions to produce a signal and thereby provideinformation about an array.
 19. The kit of claim 18, further comprisinginstructions for using said array information probe and said target toprovide information about a microarray.
 20. The kit of claim 19, whereinsaid probe is present in one or more array information elements on thesurface on an array.
 21. The kit of claim 18, wherein said instructionsinclude a protocol for spiking a sample with said target prior tocontacting said array with said sample.
 22. A system for providinginformation about an array, said system comprising: a) an arraycomprising one or more array information features; and b) a target thatspecifically binds to at least one of said one or more array informationfeatures.
 23. A method of detecting the presence of an analyte in asample, said method comprising: (a) contacting a sample suspected ofcontaining said analyte with an array of claim 1, wherein said arraycomprises a probe for said analyte; (b) detecting any resultant bindingcomplexes on the surface of said array to obtain binding complex data todetermine whether said analyte is present in said sample.
 24. The methodof claim 23, further comprising obtaining information about said arrayby assessing binding of target to said one or more array informationfeatures.
 25. The method of claim 23, wherein said analyte is a nucleicacid and said array is an array of nucleic acid probes.
 26. A methodcomprising transmitting a result obtained from a method of claim 23 froma first location to a second location.
 27. The method of claim 26,wherein said second location is a remote location.
 28. A methodcomprising receiving a result of a method of claim
 23. 29. Ahybridization assay comprising the steps of: (a) contacting at least onesample containing nucleic acids labeled with a detectable label with anucleic acid array comprising one or more array information features toproduce a hybridization pattern for said nucleic acid sample; and (b)analyzing said hybridization pattern for each detectable label toproduce data on the amounts of said target nucleic acid in said sampleand provide information about the array.
 30. A computer readable mediumcomprising programming to obtain information about an array from dataobtained using the array.
 31. A computer-readable medium comprising:information for decoding encoded array information obtained from anarray comprising one or more array information features.
 32. Thecomputer readable medium of claim 31, wherein said array is an array ofnucleic acids.
 33. The computer-readable medium of claim 31, whereinsaid information comprises a table that contains: a list of featureidentifiers; and a list of probe identifiers corresponding to saidfeature identifiers.
 34. The computer-readable medium of claim 33,wherein said table indicates that certain features of said array arearray information features.
 35. The computer readable medium of claim33, wherein said table indicates which features correspond to which bitof a code.
 36. The computer-readable medium of claim 31, wherein saidinformation indicates an executable program for decoding said encodedarray information.
 37. The computer-readable medium of claim 31, whereinsaid information is a file that has a unique identifier that correspondsto a unique identifier of an array.
 38. The computer-readable medium ofclaim 31, wherein said array information features encode binary codedinformation, and said file contains information for decoding said binarycoded information.
 39. A method for obtaining information about anarray, comprising: reading an array comprising one or more arrayinformation features to provide encoded information for said array; anddecoding said encoded information using a computer readable medium ofclaim 31 to provide information about said array.
 40. The method ofclaim 39, wherein said array is a nucleic acid array.
 41. The method ofclaim 39, wherein said scanning provides a data file comprising featureidentifiers and numerical assessments of the brightness of said arrayinformation features.
 42. The method of claim 39, wherein said decodingcomprises identifying an executable program using said file, andexecuting said program, to decode said encoded information and provideinformation about said array.
 43. The method of claim 42, wherein saidexecutable program is obtained from a remote location.
 44. A method forobtaining information about an array, comprising: encoding informationon an array using one or more array information features; and providinginformation for decoding said encoded information.
 45. The method ofclaim 44, wherein said array information is provided from a locationremote to said array.
 46. A method of assaying a sample, said methodcomprising: (a) contacting said sample with an array comprising one ormore array information features, (b) reading said array with an arrayscanner to obtain data, and (c) decoding said data using a computerreadable medium of claim 31 to provide information for said array. 47.The method according to claim 46, wherein said array is a nucleic acidarray.
 48. A method comprising transmitting a result obtained from amethod of claim 46 from a first location to a second location.
 49. Themethod of claim 48, where said second location is a remote location. 50.A method comprising receiving data representing said data obtained bythe method of claim
 46. 51. A kit for use with an array scanner, saidkit comprising: (a) a computer-readable medium according to claim 31;and (b) instructions for operating said scanner according to saidprogramming.
 52. The kit of claim 51, further comprising an array.
 53. Akit for use with an array scanner, said kit comprising: (a) an arraycomprising array information features; and (b) instructions forobtaining information for decoding encoded array information encoded bysaid array information features.